Xylem-based long-distance transport and phloem remobilization of copper in Salix integra Thunb.

Journal of Hazardous Materials 392 (2020) 122428

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Xylem-based long-distance transport and phloem remobilization of copper in Salix integra Thunb.

T

Yini Caoa, Chuanxin Mab, Hongjun Chenc, Jianfeng Zhanga, Jason C. Whiteb, Guangcai Chena,*, Baoshan Xingd a

Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou, Zhejiang, 311400, China Department of Analytical Chemistry, The Connecticut Agricultural Experiment Station, New Haven, CT, 06504, United States c Hunan Commodities Quality Supervision and Inspection Institute, Changsha, 410007, China d Stockbridge School of Agriculture, University of Massachusetts, Amherst, MA, 01003, United States b

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO

A B S T R A C T

Editor: Deyi Hou

Due to high biomass and an ability to accumulate metals, fast-growing tree species are good candidates for phytoremediation. However, little is known about the long-distance transport of heavy metals in woody plants. The present work focused on the xylem transport and phloem remobilization of copper (Cu) in Salix integra Thunb. Seedlings with 45 d preculture were grown in nutrient solutions added with 0.32 and 10 μM CuSO4 for 5 d. Micro X-ray fluorescence imaging showed the high Cu intensity in xylem tissues of both stem and root cross sections, confirming that the xylem played a vital role in Cu transport from roots to shoots. Cu was presented in both xylem sap and phloem exudate, which demonstrates the long-distance transport of Cu via both vascular tissues. Additionally, the 65Cu spiked mature leaf exported approximately 78 % 65Cu to newly emerged shoots, and approximately 22 % downward to the new roots, confirming the bidirectional transport of Cu via phloem. To our knowledge, this is the first report to characterize Cu vascular transport and remobilization in fast-growing woody plants, and the findings provide valuable mechanistic understanding for the phytoremediation of Cucontaminated soils.

Keywords: 65 Cu Translocation μ-XRF Organic component Willow

⁎

Corresponding author at: Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang District, Hangzhou, Zhejiang, 311400, China. E-mail address: [email protected] (G. Chen).

https://doi.org/10.1016/j.jhazmat.2020.122428 Received 3 November 2019; Received in revised form 12 February 2020; Accepted 28 February 2020 Available online 28 February 2020 0304-3894/ © 2020 Elsevier B.V. All rights reserved.

Journal of Hazardous Materials 392 (2020) 122428

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1. Introduction

xylem involves organic acids (Alves et al., 2011). Evidence for the longdistance transport of heavy metals associated with organic acids has also been reported for other plant species (Deng et al., 2016; Lu et al., 2013; Fu et al., 2019). These findings implied that the chemical form of the heavy metals during transport via xylem and phloem, as well as the remobilization via the phloem, will vary with metal type, metal concentration and plant species. Excessive amounts of Cu from contaminated soils can be bioaccumulated in plants and other organisms, causing toxicity and threatening the environmental safety. Thus, a series of experiments investigating xylem and phloem transport of Cu were conducted set up to further our understanding of metal accumulation and transport in fast-growing willow (S. integra). The aims of the current study were: (1) to explore the long-distance transport pattern of Cu through S. integra xylem and phloem; (2) to examine and compare the potential chelating ligands for Cu in xylem sap and phloem exudate; (3) to establish a comprehensive understanding of the Cu transport mechanism in S. integra. An understanding of the mechanisms of long-distance transport of Cu in willow species would inform and enable optimization of the rational design of technologies for the remediation of metal-contaminated soils.

Copper (Cu), as one of the essential micronutrients, and is involved in protein synthesis, photosynthesis and critical membrane activities in plants (Weinstein et al., 2011). However, Cu can also be toxic at doses beyond the optimal concentrations of approximately > 10 μM, resulting in growth inhibition, oxidative damage and nutrient loss (Cui et al., 2019; Ryan et al., 2013). Cu contamination in soils has primarily been caused by mining, smelting, fertilizer/fungicide use and waste water irrigation (Li et al., 2014; Zhao et al., 2018). Effective remediation techniques that successful manage risk for contaminated lands are in great demand worldwide. Due to the low biomass of herbaceous hyperaccumulators, fast-growing woody plants such as Salix and Populus have attracted much attention for metal phytoextraction and have shown significant potential for soil remediation (Luo et al., 2016). Previous studies have demonstrated that Salix, a non-hyperaccumulator woody plant species, can accumulate considerable amounts of heavy metals (Konlechner et al., 2013; Robinson et al., 2000; Vollenweider et al., 2006). For example, Salix caprea could accumulate 116 μg⋅ g−1 Cd and 4680 μg⋅ g−1 Zn dry weight in leaves when grown at a metal contaminated site (Unterbrunner et al., 2007). Salix integra Thunb (S. integra) could accumulate 691 μg⋅ g−1 Cu in roots, exhibiting a great potential for phytoremediation of Cu-contaminated soil via phytostabilization in wetland environment (Cao et al., 2017). Therefore, elucidation of the mechanisms of Cu transport and accumulation in willow species is of significant interest, and the findings from such studies could be used to both understand and optimize phytoremediation efficiency. Metals can be rapidly taken up by root cells, with subsequent storage in root tissues, long-distance transport via xylem and phloem, sequestration in cell walls and vacuoles, or detoxification by cytosolic metal chelation or antioxidant defense systems (Luo et al., 2016). Although several processes (e.g. metal uptake by roots, detoxification in shoot and xylem loading) have been shown to influence the efficiency of root-shoot translocation in crops or herbaceous hyperaccumulators (Ando et al., 2013; Deng et al., 2016; Zhao et al., 2018), little is known about the long-distance transport of heavy metals in non-hyperaccumulator species, particularly fast-growing woody plants. In general, metal accumulation in plants is not only associated with root uptake but also relies on internal redistribution and remobilization of the stored elements within various plant organs and tissues (Wu et al., 2010). Element redistribution in plants results primarily from transport in xylem, translocation from xylem to phloem, and remobilization through phloem transport (Taiz and Zeiger, 2010). Xylem is a direct route for metal transport from roots to shoots via water transpiration (Shen and Ma, 2001). However, element transport through the phloem (from old/mature leaves to young leaves) is more selective and active, and is a significant pathway for element allocation within plants (Erenoglu et al., 2002). Furthermore, nutritional elements are generally mobile in the phloem and can be reallocated from old/mature leaves (sources) to young tissues (sinks) (Hu et al., 2019; Page et al., 2006); however, due to the limitations of extraction and analytical methods, it is difficult to obtain this information in woody plants. Both upward and downward movement of elements have been reported in phloem transport. For example, Fismes et al. (2005) observed that in three vegetable species (bean, lettuce and radish), 63Ni was transferred throughout the entire plant following the foliar application; the metal accumulated primarily in young leaves and roots. Lu et al. (2013) reported that 68Zn could be remobilized from mature leaves to the youngest leaf tissue in Sedum alfredii Hance. As such, we speculate that Cu accumulation in new emerging tissues could be increased by phloem translocation in S. integra. During the long-distance transport via xylem and phloem, the heavy metals might be complexed with organic acids and amino acids (Ando et al., 2013). In Alyssum serpyllifolium, Ni was complexed predominantly with citric acid in the xylem sap, suggesting that metal transport via the

2. Materials and methods 2.1. Plant pre-cultivation One-year-old S. integra branches were selected from a local nursery at the Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou, Zhejiang province, China, in April 2019. Branches of S. integra with diameter of approximately 0.8 cm were cut into 12 cm in lengths, were then inserted into Styrofoam, and were pre-cultured in a 20 L plastic box containing 1/4 strength Hoagland solution without Cu as described in Cao et al. (2018) for 45 d. S. integra seedlings were maintained in a greenhouse with day/night temperature of 27 °C/20 °C and relative humidity 70–80 %. 2.2. Collection of xylem sap and phloem exudate of S. integra After 45 d of pre-culture, the seedlings were treated with 0.32 and 10 μM Cu2+ (CuSO4⋅5H2O) supplied in full-strength Hoagland solution for 5 d prior to extract xylem and phloem exudate. Seedings treated with 0.32 μM Cu2+, which was the background concentration of Cu in full strength nutrient solution, served as the control. Xylem sap was collected using an aspirator according to the method of Lu et al. (2014). The stem of each seedling was cut at approximately 3.0 cm above the junction point using sterilized scissors, and then the cut end was tightly inserted into a plastic pipette tip. The xylem sap was collected for 12 h in a 1.5 mL Eppendorf tube, which contains three calibration lines (0.5, 1 and 1.5 mL), using a vacuum pump. The volume of the sap was measured immediately after suction was completed. There were four seedlings in each treatment and the volume of the xylem sap is about 500–600 and 800–1000 μL in the control and 10 μM treatment, respectively. Then the sap was frozen at −20 °C until further analysis. Phloem exudates were collected using the EDTA-facilitated method as described in Tetyuk et al. (2013) with some modifications. Briefly, six mature leaves of each S. integra seedling were cut with the razor blade at the base of the petiole, and then the cut end of the petiole was immediately placed in the dishes containing 20 mM K2-EDTA. Subsequently, leaves were recut at the base of the petioles (about 1 mm) and the recut end was soaked into a centrifuge tube containing 1.2 mL of 20 mM K2-EDTA solution. After 1 h, the leaves were gently removed from the reaction tubes, and were washed thoroughly with deionized water to remove all EDTA. Finally, the leaves were transferred to a new centrifuge tube containing 1.2 mL of deionized water for 5 h of extraction, and the test tube was placed in a moist and dark environment to minimize the leaf transpiration. The extracted phloem exudates were stored at −20 °C until analysis. 2

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Fig. 5a. Three seedlings were treated as one replicate and four replicates were applied. At harvest, the plant was separated into labelled leaf, young leaves, mature leaves, stem, cutting and roots for the 65Cu analysis.

2.3. Organic acids determination in xylem sap and phloem exudates Organic acids in the collected xylem sap and phloem exudates were measured using ultraperformance liquid chromatography (UPLC) system with ultraviolet diode array detector (UV-DAD) (1290, Agilent Technologies, Santa Clara, CA, USA) with an ion exchange analytical column C18 (5 μm, 250 mm × 4.6 mm). The working temperature was set at 30 °C and the detection wavelength was 210 nm. The mobile phase was KH2PO4 and methanol (97:3, v/v, pH 2.75). Standard organic acids, including acetic acid, citric acid, malic acid, malonic acid, tartaric acid and oxalic acid, were run under the same condition as controls. Identification of the organic acids was performed by comparing the retention times and peak areas with the respective standard.

2.6.2. Split-root experiment After pre-culturing in Cu-free solution for 45 d, twelve seedlings were selected for the split-root experiment. The roots of each seedling were separated into two equal parts. One side of the split-root was exposed to the isotope solution of 65Cu with 10 μM 65Cu(NO3)2, and the other side was exposed to the 65Cu-free nutrient solution (Fig. 6a). After 5 d exposure, the plants were divided into roots and different aboveground tissues for the 65Cu measurement.

2.4. Synchrotron-based micro X-ray fluorescence analysis of S. integra

2.7. Determination of Cu and

After pre-culture, the S. integra seedlings were exposed to 50 μM CuSO4 for 5 d. For synchrotron-based micro X-ray fluorescence (μ-XRF) mapping, fresh petioles of leaves (young and mature leaves), stems and roots were cross-sectioned with a cryostat microtome (CryoStar NX70, Thermo Scientific, USA) at −20 °C as described in our previous study (Cao et al., 2019). Appropriate sections were selected and freeze-dried for 72 h prior to μ-XRF analysis, which was performed on the 4W1B beamline, Beijing Synchrotron Radiation Facility (BSRF, China). Twodimensional mapping was acquired by step-mode: the sample was mounted onto a precision motor-driven stage, scanning at 50 μm stepwise (with a count time of 1 s per step). The data reduction, process and mapping were carried out using PyMca package (Solé et al., 2007) and OriginPro 9.1.

Plant tissues were washed three times with deionized water. Additionally, roots were treated with 1.0 mM EDTA for 5 min to facilitate element desorption, followed by rinsing in deionized water. All the tissues were oven-dried at 70 °C for 72 h, weighed and ground into fine powder using a stainless-steel mill prior to measurement of Cu and 65 Cu tracer concentrations. The dry tissues (0.1 g) were digested in concentrated nitric acid (HNO3) and hydrogen peroxide (H2O2) (4:1, v/ v) using a hot block system (ED36, Lab Tech, Germany). The digestion was diluted to 25 mL with 2 % (v/v) diluted HNO3 solution. For samples of xylem sap and phloem exudates, a subsample of 0.2 mL was mixed with 10 mL of 2 % (w/v) HNO3 (Lu et al., 2008). The total concentration of Cu and other nutritional elements were determined using inductively coupled plasma atomic emission spectrometry (ICPAES, Perkin Elmer Optima 8000, Waltham, USA). Poplar leaves were used as the certified reference material (GBW 07604, National Research Center for Certified Reference Materials, China) to ensure the accuracy of the elemental analysis throughout the plant digestion. Good agreement was obtained between our method and certified values with the accuracy of 96 ± 2.2 %. The ratio of 65Cu/63Cu (Rfin) in S. integra was obtained directly from the analysis of by ICP mass spectrometry (Agilent 7700x, California, USA). The amount of 65Cu in the different tissues was calculated from the equation based on the isotope ratio. Newly accumulated 65Cu concentrations in different tissues of S. integra (Cuacc, mg ∙ kg−1 DW) were calculated from the total Cu concentration (Cutot, mg⋅ kg−1 DW) in the respective plant tissues, according to Eq. (1) (Wu et al., 2010)

2.5. Re-rooting experiment–redistribution of Cu in S. integra After 45 d pre-cultivation, 30 seedlings were selected and grown in full-strength Hoagland solution containing 0.32 (control) and 10 μM CuSO4 for 5 d. Afterwards, the whole roots of S. integra were excised and part of the leaves were removed, retaining fully expanded mature leaves (approximately eight) and the seedling apexes (with four to six young leaves) in order to clearly distinguish the “sources” and “sinks” (Fig. 3a). The seedlings without roots were then washed with deionized water and kept in deionized water in darkness overnight for wound healing. The seedlings were then transferred into the Cu-free nutrient solution and re-cultured for 28 d to generate new roots. Before and after re-rooting, the seedlings were divided into newly emerged leaves, original young leaves, original mature leaves, stems, cuttings, original roots and newly emerged roots (Fig. 3a). After incubation, the tissues were harvested for Cu measurement. In a separate experiment, 45 d-old S. integra seedlings were grown in nutrient solution amended with 0.32 and 10 μM CuSO4 for 5 d. To investigate the influence of senescence on Cu remobilization, the retained mature leaves of S. integra were randomly divided into two equal parts, one of which was covered with aluminum foil. Differently treated leaf tissues were harvested after 28 d re-rooting for Cu measurement. 2.6. Foliar application and split-root experiment with

65

Rfin =

65

Cu tracer concentration

(Cuacc × f 65-enr )+f 65-nat(Cutot − Cuacc) (Cuacc × f 63-enr )+f 63-nat(Cutot − Cuacc)

(1)

which derived the formula for Cuacc

Cuacc =

Cutot[f65-nat − (Rfin × f 63-nat)] Rfin (f 63-enr − f 63-nat) + f 65-nat − f65-enr

(2) 65

63

where f65-nat and f63-nat are the nature abundance of Cu and Cu in normal nutrient solution (30.83 % and 69.17 %, respectively); f65-enr and f63-enr are the abundances of 65Cu and 63Cu in 65Cu-enriched Cu (NO3)2 bought from SPEX (99.26 % and 0.74 %, respectively). All data of 65Cu in the figures refer to Cuacc only, which do not include the 65Cu accumulated with “normal Cu” (Cutot-Cuacc).

Cu

2.6.1. Foliar application The Cu stable isotope−65Cu was chosen as the Cu2+ source to avoid the contamination from other sources. A standard volume of 10 μg⋅ mL−1 65Cu(NO3)2 (65Cu abundance: 0.9926, SPEX Certiprep, USA) solution was heated at 75 ℃ and concentrated to 1 mM according to Lu et al. (2013). The pH of the solution was adjusted to 5.8 using 5% KOH. One fully expanded mature leaf was selected as the labelled leaf for each seedling after pre-culturing in Cu-free solution for 45 d. To facilitate the 65Cu uptake, each labelled leaf was cut approximately 2 mm from the leaf tips. The tips, about 1/5 of the leaf area, were then soaked in 1 mL of labelling solution (1 mM 65Cu(NO3)2) for 5 d as shown in

2.8. Statistical analysis All data were statistically analyzed using the Data Processing System Version software (DPS13.01, Zhejiang University, Hangzhou, China), and the graphical work was performed by OriginLab software (OriginPro 9.1). Prior to statistical analysis, all data were tested to confirm the normality by Shapiro-Wilk procedure and Levene’s Test was used to test the null hypothesis that variances are homogeneous. Statistical significance between the two treatments (control and 10 μM levels) for all data were assessed using Student’s t test (P < 0.05). For 3

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the different Cu concentrations in different tissues (leaves, stems, cuttings, roots), one-way analysis of variance (ANOVA) was performed on the datasets, and the mean and standard deviation (SD) of each treatment, as well as the least significant difference (LSD) (P < 0.05 and P < 0.01), were calculated. The translocation factors (TFs) of Cu from root to leaf were calculated by using Cu concentrations according to Shi and Cai (2009). The bioaccumulation factor (BAF) were used to indicate the efficiency of S. integra in accumulating Cu into its tissues from the surrounding environment (Ladislas et al., 2012; Zacchini et al., 2009).

TF=

LeafCutot RootCutot

BAF=

Table 2 Organic acid composition in xylem sap and phloem exudate extraction of S. integra ‘Yizhibi’ exposed to 0.32 (control) and 10 μM Cu2+ (CuSO4 ∙ 5H2O) for 5 d. Organic acid content (mg∙L−1)

Tartaric acid Fumaric acid Acetic acid Citric acid Malic acid

(3)

Charvest tissues Csolution

(4)

Xylem sap

Phloem exudate extraction

Cu levels (μM)

Cu levels (μM)

control

10

control

10

9.69± 0.55 13.31±0.03 27.44±1.64 48.85±0.95 316.08± 1.32

8.56±0.26* 25.15± 0.01** 35.58± 0.88* 104.37± 0.57** 574.88± 1.71**

8.50± 0.10 0.07± 0.01 5.46± 0.76

7.06± 0.09** 0.06± 0.02ns 5.42± 0.60ns − −

−

Values represented means ± SD (n = 3). Asterisks (* and **) represented significant differences of P < 0.05 and P < 0.01, respectively, across both Cu treatments.

where Charvest tissues is Cu concentration in harvest tissue and Csolution is Cu concentration in the solution.

tartaric acid concentration by 11.7 % as compared to the control, while increases in the levels of other four organic acids in the xylem sap were evident with Cu exposure. Different patterns in the levels of organic acids in the phloem exudate were found. No citric acid and malic acid were detected in the phloem exudate of mature leaves (Table 2). Fumaric acid was found at a relatively low concentration (< 0.1 mg⋅L−1) in the phloem exudate, suggesting this acid likely played a minor role in Cu chelation during the phloem transportation in S. integra. In the Cu treatment, the concentration of tartaric acid was decreased by 16.9 % in the phloem exudates, but levels of fumaric and acetic acid were not impacted on Cu exposure. Additionally, a correlation analysis indicated that all the five organic acids were highly correlated with the Cu concentration in the xylem sap. Conversely, in the phloem exudate only malic acid was closely associated with Cu content (Table S1).

3. Results 3.1. Cu and other nutrient elements in the xylem sap and phloem exudates The Cu concentration in the xylem sap of S. integra was 3.25 and 6.32 μM in the control and 10 μM Cu treatment, respectively (Table 1). However, the Cu concentration in the phloem exudate was between 0.11–0.23 μM. Significant differences within the Cu treatments (P < 0.01) were observed in both the xylem sap and phloem exudate. The Cu concentration in the xylem sap and phloem exudates in the 10 μM Cu treatment was 0.94 and 1.09-fold higher than the respective control. Exposure to 10 μM Cu significantly decreased the Mn concentration by 74.7 % in xylem sap as compared to the control. A negative correlation between the concentration of Cu and Ca in the xylem sap was evident; a positive correlation between Cu and other elements (B, Mg, Na and Mo) was also found in the xylem sap (Fig S1). However, in the phloem exudates Cu was negatively correlated with B, Mg, Na and Mo.

3.3. Spatial imaging of Cu in the cross sections of petioles and stems The cross sections of petioles (young and mature leaf) and stems were evaluated by μ-XRF imaging after 5-d exposure to 50 μM Cu (Fig. 1). In the petiole of mature leaves, high Cu signals were detected in the vascular bundles, while the Cu intensity in the mesophyll and epidermis were notably lower. However, in the petioles of young leaves the Cu intensity in the vascular bundles was relatively low, and only few high intensity Cu spots were observed in the mesophyll close to the epidermis. Thus, the Cu accumulation in the petiole cross sections demonstrate an age-specific variation in distribution patterns in S. integra. Additionally, significant amounts of Cu in the peripheral region of the

3.2. Organic acids in the xylem sap and phloem exudate Five organic acids were detected in the xylem sap, with a descending order in concentrations of malic acid > citric acid > acetic acid > fumaric acid > tartaric acid (Table 2), regardless of Cu exposure. Malic acid was the predominant organic acid in the xylem sap; the concentration was 574.9 mg⋅ L−1 in the 10 μM Cu treatment, which was 0.82-fold higher than the control. Exposure to Cu decreased the

Table 1 The concentration of Cu and other nutritional elements in xylem sap and phloem exudate of S. integra ‘Yizhibi’ upon exposure to 0.32 (control) and 10 μM Cu2+ (CuSO4) for 5 d. Element concentrations (μM)

Cu Fe Mn Zn Ca K P B Mg Na Mo

Xylem sap

Phloem exudate extraction

Cu levels (μM)

Cu levels (μM)

Control

10

control

10

3.25± 0.23b 19.34±0.25b 12.99±1.09a 2.32± 0.08b 1048.62± 106.79a 5510.90± 472.09a 532.13± 42.99b 25.70±1.67b 390.59± 34.26a 143.83± 8.34b 0.12± 0.00b

6.32±0.21a 34.62±0.24a 3.29±0.21b 13.68±1.96a 671.69± 43.82b 6558.97± 104.18a 685.97± 21.24a 49.91±5.83a 498.11± 63.76a 244.17± 8.54a 0.87±0.12a

0.11±0.01b 4.88±0.24a 0.66±0.02a 1.69±0.20a 19.24± 1.42b 343.67± 0.33a 4.92±0.13b 10.98± 0.09a 9.90±0.78a 9.54±0.41a 0.19±0.01a

0.23±0.02a 3.71±0.20b 0.59±0.02b 1.78±0.04a 44.22± 3.10a 385.50± 14.95a 6.23±0.15a 8.27±0.06b 5.01±0.29b 7.29±0.31b 0.15±0.00b

Note: Values represent means ± SD (n = 3). Distinct letters in the same row indicate significant different between control and 10 μM levels according to Student’s t test (P < 0.05). 4

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Fig. 1. μ-XRF images of Cu in cross sections of petioles ((a) young leaf (b) mature leaf (c) stem of S. integra exposed to 50 μM Cu for 5 d. Ep, epidermis; M, mesophyll; VB, vascular bundles.

3.5. Cu accumulation and remobilization to newly emerged tissues

cylinder were observed in the stem cross section, where the xylem tissues were localized, and the Cu intensity became markedly lower in the epidermal and cortex tissues.

After exposed to 0.32 (control) and 10 μM CuSO4 for 5 d, all roots of S. integra were excised and re-cultured in nutrient solutions without Cu to generate newly emerged tissues (Fig. 3a). The Cu concentrations in different tissues (original young leaves, original mature leaves and roots) of S. integra varied significantly after 5-d Cu exposure. Cu at 40.6 and 873.5 mg⋅ kg−1 was found in the control and Cu treated roots (Fig. 3b). Newly emerged roots were observed at Day 4 after removing the whole original roots from S. integra. After 28-d re-rooting, the Cu concentrations of the newly emerged roots were 13.3 and 16.8 mg⋅ kg−1 (P < 0.05) in the control and 10 μM Cu treatment, respectively. The Cu concentrations in the newly emerged leaves were 12.4 and 16.7 mg⋅ kg−1 in the control and 10 μM Cu treatment, respectively (P < 0.05). However, a significant decrease in the Cu concentration was evident in the original young leaves and mature leaves (except for the original young leaves with 10 μM Cu treatment) when compared with the respective treatment before re-rooting (Fig. 3b). More specifically, approximately 15–17 % Cu stored in the mature leaves of S. integra was exported for remobilization. The Cu concentrations in the covered original mature leaves was markedly reduced as compared to the

3.4. Spatial imaging of Cu treated roots The μ-XRF images of the root cross section, root tips (0−1 cm), root segment (> 5 cm) and lateral root show the localization of Cu in S. integra roots (Fig. 2). In the 50 μM Cu treatment, significant Cu intensity was evident in the cortex, and relatively high Cu signals were also observed in the cylinder regions near the periphery, most likely corresponding to the xylem tissues where transport of metals from roots to shoots occurs (Fig. 2a). The distribution of Cu in the root tips exhibited preferential accumulation in the root apex, particularly at the meristematic elongation zone, but low intensity was observed at the epidermis of the root cap (Fig. 2b). Additionally, high Cu levels were also noted in the elongation zone, although the Cu content was much lower than that of meristematic zone. In the mature root segment (> 5 cm), Cu was primarily localized in the root stele and relatively high Cu content were also detected at the junction where the lateral and primary root stele are connected. 5

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uncovered original mature leaves (P < 0.01) (Fig. 4), indicating that dark-induced senescence in the mature leaves of S. integra could increase the Cu remobilization out of these tissues. 3.6. Foliar-applied

65

Cu and split-root experiment

Approximately 87 % of the applied 65Cu was accumulated in the labelled mature leaf of S. integra (Fig. 5b). Although very little 65Cu was found in the non-labelled tissues, there were significant differences among those non-labelled tissues (P < 0.01). In particular, the labelled Cu was re-allocated to the old leaves, roots and young leaves. Most of the 65Cu (78 %) exported from the spiked mature leaf was translocated to the above-ground tissues, and approximately 22 % was moved downward to the roots (Table S2). In split-root experiment with willow seedlings exposed to 65Cu, the 65 Cu concentration of the directly exposed roots was markedly higher than that on the non-65Cu treated side (Fig. 6b); only 6.99 mg⋅ kg−1 65 Cu was found on the non-65Cu treated side. In addition, the 65Cu concentration of above-ground tissues were also significantly lower compared to the roots on the non-65Cu treated side (P < 0.05). These results suggest that about 72 % of the 65Cu content moved upward to the shoots, and of that, 22 % of the 65Cu was transferred to the leaves (Table S3). 4. Discussion

Fig. 2. μ-XRF images of Cu in (a) the cross section of root tip and (b) the whole root tip (0-1 cm), root segment (> 5 cm) and lateral root zone of S. integra exposed to 50 μM Cu for 5 d. Ep, epidermis.

4.1. Cu transport via xylem in S. integra In the present study, the Cu concentration in root was markedly Fig. 3. The re-rooting experiment was conducted as shown in (a). Intact seedings of S. integra ‘Yizhibi’ were pre-treated in the nutrient solution containing 0.32 and 10 μM Cu2+ (CuSO4⋅5H2O) for 5 d, then all roots and partial leaves were excised and kept only the youngest tissues on the top and eight mature leaves. The seedlings were re-cultured in the nutrient solution without Cu for 28 d. Different tissues of S. integra ‘Yizhibi’ were harvested before and after re-rooting. Cu concentrations (b) in different tissues of S. integra ‘Yizhibi’ upon exposure to nutrient solution containing 0.32 and 10 μM Cu2+ (CuSO4⋅5H2O) for 5 d and re-cultured in the nutrient solution without Cu for 28 d. Bars represent means ± SD (n = 5), and single (*) and double asterisks (**) represent significant differences at P < 0.05 and P < 0.01, respectively, in Cu accumulations within the same tissue before and after rerooting.

6

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Fig. 4. Effect of dark-induced senescence on Cu redistribution in S. integra ‘Yizhibi’. Bars represent means ± SD (n = 4), and asterisks represent significant differences in Cu accumulations between the two parts of the original mature leaves at P < 0.05. Fig. 5. Copper remobilization from labelled 65 Cu leaf to other tissues in S. integra ‘Yizhibi’. (a) Scheme of the labelled 65Cu leaf exposure experiment. A fully expanded mature leaf of S. integra ‘Yizhibi’ was labelled with 65Cu (1 mM) for 5 d, and different leaf (youngest, mature, and old) tissues were harvested for 65Cu analysis. (b) 65Cu concentrations in different plant tissues of S. integra ‘Yizhibi’. Bars represent means ± SD (n = 4).

Fig. 6. 65Cu accumulation (mg⋅kg−1 DW) in roots, cutting, stem and leaves in seedlings of S. integra ‘Yizhibi’ in the root splitting experiment. One side of roots was exposed to 10 μM Cu (+ 65Cu) and the other root side without Cu (− 65Cu). Bars represent means ± SD (n = 4).

plant species (Arduini et al., 1996; Liao et al., 2000). Additionally, the high retention of Cu in the roots could disturb the balance of plant hormones, limiting the xylem-mediated transport of Cu to mature leaves (Ando et al., 2013). Thus, further transport via the phloem to the growing young leaves is highly regulated in order to maintain the Cu homeostasis in young leaves. The variations of Cu distribution between roots and above-ground tissues might does vary with plant species,

higher than that in above-ground tissues after the 5-d exposure period (Fig. 3b), which is in line with previous results demonstrating that Cu preferentially localizes in the roots (Cao et al., 2019; Liao et al., 2000; Zhao et al., 2018). The significant decrease of TF and BAF was observed as solution Cu level elevated, and the considerable amount of Cu retained in the roots of S. integra suggests rate limited translocation to above-ground tissues (Table S4), which has also been reported in other 7

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could be a useful evidence for evaluating the significance of Cu transport in phloem. Similarly, Zn and Ni enrichment in the phloem exudates were found in hyperaccumulators such as Sedum alfredii and Noccaea caerulescens (Deng et al., 2016; Lu et al., 2013), suggesting that these species have an inherent high phloem loading ability for heavy metals. In the re-rooting experiment, Cu accumulation was also found in the newly emerged roots growing in the Cu-free nutrient solution for 28 d (Fig. 3b). Since the seedlings of S. integra were cultured in the nutrient solution without Cu, the upward transportation of xylem appears to not be the primary distribution pathway. 65Cu was detected in the nonlabelled leaves and roots, although the concentrations of 65Cu were relatively low. Both the re-rooting and Cu isotope exposure experiments demonstrate that Cu was transported not only from root-to-shoot but also from shoot-to-root. The bidirectional transport of other heavy metals (Cd, Zn, Al and Ni) via the phloem has also been documented in white lupin (Page et al., 2006), Sedum alfredii (Lu et al., 2013), Camellia oleifera (Zeng et al., 2013) and Noccaea caerulescens (Deng et al., 2016). Xylem transport is essentially unidirectional, whereas phloem transport is bidirectional; additionally, solute mobilization to roots is more or less an evidence against exclusive translocation in the xylem (Taiz and Zeiger, 2010). As such, Cu accumulation in newly emerged roots implies the metal transport from leaves to roots via the phloem (Fig. 3b), although Cu amounts in the phloem exudates are quite low. The positive correlation between Cu concentrations in newly emerged roots and leaves (r2 = 0.95, P < 0.01) suggests that the higher the Cu concentration in the aerial tissues, the greater the amount transported to the newly emerged roots. The newly emerged roots are well connected with the cuttings and originate from the secondary phloem or pitch, cambium and vascular bundles of new roots; this is also beneficial for the bidirectional transport of elements (Zeng et al., 2013). In general, element remobilization from old/mature organs to young organs occurs via phloem (Hu et al., 2019). Therefore, the redistribution of Cu between young leaves and mature leaves directly demonstrates Cu remobilization via phloem in S. integra. To our knowledge, this is the first report to reveal this process in a fast-growing woody species such as S. integra, and these findings further our understanding of Cu accumulation dynamics in terrestrial plants.

growth stage of the life cycle and environmental conditions (Lombi et al., 2000). Heavy metals are primarily transported to the aboveground tissues via xylem (Clemens et al., 2002; Wu et al., 2010). The efficiency of metal transport from roots to shoots relies on several processes, including symplastic uptake, root sequestration, xylem loading and foliar uptake (Lu et al., 2013). The μ-XRF image from the current study show that Cu was preferentially localized in the root stele and vascular bundles (cross sections of root, stem and petioles) within S. integra (Figs. 1 and 2b). These results are likely indicative of the xylem loading of Cu for long-distance translocation to the aerial tissues. In our study, the Cu concentration in the xylem sap was greatly affected by the Cu dose in the nutrient solution (Table 1). Additionally, the Cu concentration in S. integra xylem sap and the stem were highly correlated (r2 = 0.97, P < 0.01). The correlation analysis indicates that the higher Cu concentrations in the xylem sap resulted in higher Cu amounts in the stem, which might subsequently control the efficiency of Cu transport via xylem to the above-ground tissues. Previous studies have also demonstrated that the long-distance transport of heavy metals occurred primarily in the xylem and was driven by transpiration (Konrad et al., 2019; Shen and Ma, 2001). According to the results of partial transpiration suppression on Cu accumulation in leaves after 1 h, the leaf transpiration rate was significantly reduced from 3.40 to 3.06 mmol m−2s-1 (Table S5). After inhibition for 48 h, the S. integra leaf Cu concentration decreased from 18.25 to 17.75 mg⋅ kg-1, clearly demonstrating that suppression of xylem transport could significantly reduce the Cu accumulation in the leaves. However, high metal accumulation in leaves may not be always a function of high leaf transpiration rates and the efficiency of element accumulation in the aerial tissues may vary with seasons of the year and other environmental factors (Zeng et al., 2011). The split-root system is a valuable method to investigate the local and systemic regulation mechanisms in plants as affected by environmental stressors, such as heavy metal exposure (Ma et al., 2017; Wang et al., 2012). The great advantage of this system is that the root system was separated into two parts and placed in two independent containers, but could share the same aerial tissues (Larrainzar et al., 2014). If the untreated part of the root system exhibits a response when a stimulus is applied to the treated part, the long-distance transport and systemic regulation are suggested to be involved in (Ma et al., 2017). After 5 d of 65 Cu exposure, 65Cu was detected in the non-65Cu treated leaves, suggesting efficient root to shoot transport was involved in S. integra (Fig. 6). The 65Cu concentration of above-ground tissues was significantly lower than roots, indicating that the Cu transport could be somewhat inhibited in the split root treatment. Thus, the results of 65Cu analysis in the above-ground tissues and the overall Cu concentration in xylem sap both suggest efficient element transport in the xylem. The Cu concentrations in leaves were clearly affected by transpiration rate, and the xylem mediated the long-distance transport of Cu from roots to leaves. In addition, we also observed the phenomenon of homolateral 65 Cu transport in S. integra as a result of the significant difference of root 65Cu concentrations between the 65Cu and non-65Cu treated side. These results point out that Cu can also be transported via the phloem, particularly when the leaf transpiration rate is low, and that both types of vascular tissues are important for Cu translocation.

4.3. Organic ligands involved in Cu transportation Chelation is known to play a key role in metal phytoextraction, and the stability of the metal-chelate complex in plants directly affects the transport efficiency (Vítková et al., 2015; Zhao et al., 2018). Previous studies have reported that metals in plants are mostly associated with organic ligands, such as histidine, citrate and malate (Ghnaya et al., 2013; Lu et al., 2013). In this study, the concentrations of organic acids were significantly higher than amino acids in the xylem sap of S. integra (Tables 2 and S6). Citric and malic acids were the two analytes present at the high concentration in the xylem sap, implying that both are important to the xylem transport of Cu in S. integra. Similarly, Lu et al. (2013) reported that citrate participated in Zn transport via the xylem in Sedum alfredii, and Fu et al. (2019) demonstrated that citrate was involved in the xylem transport of Cd in Oryza sativa. Additionally, malate was also important in the xylem transport of Cd in Brassica juncea (Wei et al., 2007). Although a small portion of Cd could be complexed with citric acid, most of the metal was still in the free ionic form in the xylem sap of the Arabidopsis helleri (Ueno et al., 2008). It is possible that the hyperaccumulator was less sensitive to Cd in the xylem vasculature, and that binding with organic ligands may be unnecessary for Cd translocation (Fu et al., 2019). Notably, differences in plant species, metal type and dose can result in the contradictory findings. Ryan et al. (2013) reported that the mechanism of Cu translocation was different between strategy I plants (dicotyledons and nongraminaceous monocotyledons) and strategy Ⅱ plants (graminaceous monocotyledons), and that dicots had higher concentration of pectin and more binding sites than do monocots (Guigues et al., 2016),

4.2. Cu remobilization via phloem in S. integra In an intact plant system, it is difficult to investigate long-distance phloem transport as only few techniques are suitable and phloem is particularly sensitive to wounding (Peuke et al., 2006; Zeng et al., 2013). In the present study, mature leaves were collected to extract phloem exudate, and the direct measurement of the Cu content in phloem exudates also made it possible to compare the differences between the control and the Cu treatments. The Cu concentrations in phloem exudates in the mature leaves were 0.11–0.23 μM (Table 1), indicating that the direct measurement of Cu concentration in this fluid 8

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Fig. 7. A conceptual model for long distance transport of copper in fast-growing woody plant S. integra ‘Yizhibi’.

whereas young leaves accumulate only minor amounts of the metal. Moreover, metal transport in the xylem is usually presented as free hydration cations (Centofanti et al., 2013), and finally, citrate and malate seem to play a key role in xylem transport of Cu. (3) When xylem flow reaches minor veins and fills in the apoplastic spaces in leaves, heavy metal ions could be unloaded into the leaf symplast, which is a primary storage site for heavy metals (Milner and Kochian, 2008). (4) Phloem transport can be either root-to-shoot or shoot-to-root, relying on the relative balance of sources and sinks (Deng et al., 2016). Specifically, the movement of metals in the phloem system is mainly driven by osmotic pressure generated by the concentration gradient of photosynthate between sources and sinks (Taiz and Zeiger et al., 2010). In addition to the upward movement of metals, plants can also allocate and remobilize metals from mature leaves to the youngest developing leaves (Hu et al., 2019; Lu et al., 2013), with a fraction of the metals being originally transported through the xylem. (5) Phloem companion cells containing metal-abundant apoplastic fluid could utilize a number of metal influx transporters in the cell membranes, and further accumulate metal ions into the cytosol (Deng et al., 2016). Specifically, some significant metal ions could be chelated by various organic molecules (i.e. organic acids or amino acids), and then the metal-ligand complexes are transferred from companion cells to phloem vessels via plasmodesmata (Taiz and Zeiger et al., 2010).

consequently altering Cu transport. For non-hyperaccumulator species, metals in the phloem exudates are usually present at low concentrations. Accordingly, in the current study, a relatively low concentration of Cu in the phloem exudate was detected. It is worth noting that a small number of organic acids at low concentrations were detected in the phloem exudate (Table 2). The content of fumaric acid and acetic acid did not exhibit significant difference between the control and 10 μM Cu treatment, indicating that these two organic ligands are of less importance to the phloem transport of Cu. However, the tartaric acid content exhibited significant difference with Cu exposure, indicating this organic acid is a likely chelator involved in Cu phloem transport. 4.4. Conceptual model for long-distance transport of Cu in S. integra A conceptual model for long-distance transport of Cu in S. integra is shown in Fig. 7. (1) S. integra roots exhibit a high affinity to absorb the free Cu in solutions. Cu is predominately localized in root apoplast, which results in relatively low amount of Cu translocation to the root stele. These results are in accordance with the observation of Cu accumulation in meristem of the root tip, as well as the primary and lateral root conjunction (Fig. 2). Generally, the root stele and lateral root zone were limited by the pericycle cell layer, where the Casparian strip is inherently incomplete and inhibits the radial movement of Cu in the apoplastic pathway (Lu et al., 2017; Zhao et al., 2018). It has been demonstrated that heavy metals can be efficiently moved through the root symplast and loaded into xylem vessels, where subsequent transport to the above-ground tissues is driven by transpiration stream (Deng et al., 2016; Zeng et al., 2013). The movement of heavy metals into the symplastic path way was controlled by the expression of several efflux transporters, such as Cation Diffusion Facilitators (CDFs), Heavy Metal ATPases (HMAs) family members, Zinc/Iron regulated Proteins (ZIPs) and Natural Resistance And Macrophage Proteins (NRAMPs) (Luo et al., 2016). (2) Cu transport to the above-ground tissues of S. integra occurs via the xylem flow, with most of the metal being finally stored in mature leaves as the result of transpiration stream and root pressure,

5. Conclusions The current study clearly demonstrated the xylem transport and phloem remobilization of Cu in shrub willow S. integra by using multiple advanced methods. High level of Cu in xylem sap and high Cu intensity in μ-XRF in xylem tissues directly confirm the significance of xylem transport of Cu from roots to shoots. Differential spatial distribution of Cu in root apex and lateral zone indicates that Cu was more preferred to transport to root stele. The re-rooting and stable isotope 65Cu trace experiment pointed out the bidirectional transport of Cu via phloem. The Cu remobilized from old/mature leaves to newly growing leaves in S. 9

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integra, which might be attributed to the high Cu requirement of the growing leaves. Cu stress markedly increased the contents of the organic acids contents in xylem sap but decreased the phloem exudate. Mechanisms involved in long-distance transport of Cu in shrub willows will be of great significance, which is not only essential to understand the Cu tolerance and its potential capacity but also for improving the soil remediation strategies.

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Credit author statement Dr. Yini Cao: Conceptualization, investigation, data analysis, writing the paper; Dr. Chuanxin Ma: data analysis and writing the paper; Mr. Hongjun Chen and Dr. Jianfeng Zhang: Sample preparation, determination and data analysis; Dr. Guangcai Chen: Conceptualization, investigation, manuscript revision, Funding acquisition and project administration; Drs. Jason C. White and Baoshan Xing: Manuscript comments and revision. Declaration of Competing Interest The authors declared that they have no conflicts of interest to this work. We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled. Acknowledgements This work was supported by the Fundament Research Funds of CAF (Grant No. CAFYBB2019SZ001) and National Natural Science Foundation of China (Grant No.31470619). The μ-XRF analysis of this research was carried out at the 4W1B beamline of Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences. The authors sincerely acknowledge Drs. Dongliang Chen, Juncai Dong and all staff members of 4W1B, for their support in measurements and data reduction. The authors also thank Dr. Junkun Lu from RITFCAF, for his advice on sap extraction. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jhazmat.2020.122428. References Alves, S., Nabais, C., Simoes, G.M.L., Correia, D.S.M., 2011. Nickel speciation in the xylem sap of the hyperaccumulator Alyssum serpyllifolium ssp. lusitanicum growing on serpentine soils of northeast Portugal. J. Plant Physiol. 168, 1715–1722. Ando, Y., Nagata, S., Yanagisawa, S., Yoneyama, T., 2013. Copper in xylem and phloem saps from rice (Oryza sativa): the effect of moderate copper concentrations in the growth medium on the accumulation of five essential metals and a speciation analysis of copper-containing compounds. Funct. Plant Biol. 40, 89–100. Arduini, I., Godbold, D.L., Onnis, A., 1996. Cadmium and copper uptake and distribution in Mediterranean tree seedlings. Physiol. Plant. 97, 111–117. Cao, Y.N., Ma, C.X., Chen, G.C., Zhang, J.F., Xing, B.S., 2017. Physiological and biochemical responses of Salix integra Thunb. under copper stress as affected by soil flooding. Environ. Pollut. 225, 644–653. Cao, Y.N., Ying, Z., Ma, C.X., Li, H.M., Zhang, J.F., Chen, G.C., 2018. Growth, physiological responses, and copper accumulation in seven willow species exposed to Cu—a hydroponic experiment. Environ. Sci. Pollut. R. 25, 19875–19886. Cao, Y.N., Ma, C.X., Zhang, J.F., Wang, S.F., White, J.C., Chen, G.C., Xing, B.S., 2019. Accumulation and spatial distribution of copper and nutrients in willow as affected by soil flooding: a synchrotron-based X-ray fluorescence study. Environ. Pollut. 246, 980–989. Centofanti, T., Sayers, Z., Cabello-Conejo, M., Kidd, P., Nishizawa, N., Kakei, Y., Davis, A., Sicher, R., Chaney, R., 2013. Xylem exudate composition and root-to-shoot nickel translocation in Alyssum species. Plant Soil 373, 59–75. Clemens, S., Palmgren, M.G., Krämer, U., 2002. A long way ahead: understanding and engineering plant metal accumulation. Trends Plant Sci. 7, 309–315.

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