Differences in uptake and accumulation of copper and zinc by Salix clones under flooded versus non-flooded conditions

Differences in uptake and accumulation of copper and zinc by Salix clones under flooded versus non-flooded conditions

Chemosphere 241 (2020) 125059 Contents lists available at ScienceDirect Chemosphere journal homepage: www.elsevier.com/locate/chemosphere Differenc...

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Chemosphere 241 (2020) 125059

Contents lists available at ScienceDirect

Chemosphere journal homepage: www.elsevier.com/locate/chemosphere

Differences in uptake and accumulation of copper and zinc by Salix clones under flooded versus non-flooded conditions Weidong Yang a, b, Fengliang Zhao a, *, Yuyan Wang c, Zheli Ding d, Xiaoe Yang b, Zhiqiang Zhu e a

Ministry of Agriculture and Rural Affairs Danzhou Scientific Observing and Experimental Station of Agro-Environment, Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences (CATAS), Haikou, 571101, China Ministry of Education Key Laboratory of Environmental Remediation and Ecological Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou, 310058, China c Poyang Lake Eco-economy Research Center, Jiujiang University, Jiujiang, China d Haikou Experimental Station, Chinese Academy of Tropical Agricultural Sciences (CATAS), Haikou, 571101, China e Institute of Tropical Agriculture and Forestry, Hainan University, Renmin Road 58, Haikou, 570228, China b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 The Salix clones showed wide variation in biomass production and accumulation of Cu/Zn.  Hypertrophied lenticels and adventitious roots conferred tolerance to flooding.  Non-flooded Salix clones exhibited good phytoextraction capacities of Cu/Zn.  Flooded Salix clones showed phytostabilization potentials of Cu/Zn, especially for Cu.  EDTA was suitable for predicting bioavailability of Cu and Zn under current condition.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 June 2019 Received in revised form 27 September 2019 Accepted 3 October 2019 Available online 4 October 2019

The climate-driven flooding poses a challenge for phytoremediation of contaminated soil, and the willow (Salix spp.) is a promising candidate coping with climate change and environmental pollution. In this study, uptake and accumulation of copper (Cu), zinc (Zn) and their bioavailability in the rhizosphere across the Salix clones under flooded versus non-flooded (control) conditions were investigated using a pot experiment. The tested Salix clones grew well without showing any toxic symptoms under nonflooded soil condition; in contrast, the clones showed 100% survival for long-term flooding with the development of hypertrophied lenticels and adventitious roots. There were wide clonal variations in biomass production and accumulation of Cu and Zn under flooded and non-flooded conditions. Flooded treatments dramatically decreased aboveground biomass across the Salix clones to different extents compared to the control. The non-flooded clones exhibited relatively high accumulation capacities of Cu and Zn in aerial parts. However, the flooded clones resulted in more substantial reductions in Cu and Zn accumulation in aerial parts, and most of Cu and Zn were limited in roots. EDTA-extractable Cu and Zn predicted well bioavailability of Cu and Zn to the Salix clones under the current condition. It was concluded that the Salix clones exhibited Cu and Zn phytoextraction traits (non-flooding) or

Handling Editor: T Cutright Keywords: Salix spp. Copper Zinc Flooding Phytoremediation

* Corresponding author. E-mail address: [email protected] (F. Zhao). https://doi.org/10.1016/j.chemosphere.2019.125059 0045-6535/© 2019 Elsevier Ltd. All rights reserved.

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phytostabilization traits (flooding), which provides a valuable insight into phytomanagement of contaminated soils by willows subjected to flooding stress. © 2019 Elsevier Ltd. All rights reserved.

1. Introduction In the past decades, climate change and heavy metal contamination have become two severe issues around the world as a result of anthropogenic activities. By 2030, a predicted 30% increase in heavy precipitation caused by climate changes indicates that flooding stress will be more severe in the future (Valliyodan et al., 2016), and soil flooding affects >1700 Mha of land worldwide every year (Striker and Colmer, 2016). Meanwhile, the contamination of copper (Cu) and zinc (Zn) poses a significant threat to plants, animals and humans, although Cu and Zn are essential elements for them (Song et al., 2004; MacFarlane et al., 2007; Padmavathiamma and Li, 2010). Cu or Zn pollution results from human activities, such as mining and refining, leather tanning, textile and microelectronics, chemical fertilities, manures, and pesticides (Song et al., 2004; MacFarlane et al., 2007; Yang et al., 2014). Phytoremediation of contaminated soils is defined as “use of plants or their rhizosphere to remove contaminants through harvestable sections including stems and leaves (phytoextraction) or control and immobilize contaminants in roots (phytostabilization)”, which is a cost-effective and environmentally friendly alternative to traditional remediation technologies, especially for large areas (Marques et al., 2007; Padmavathiamma and Li, 2010). Phytoremediation potential of a plant is primarily determined by tissue metal concentrations, biomass production and environmental factors (Jensen et al., 2009). The aerobic environment condition benefits plant growth to increase phytoremediation efficiency. However, the frequent regional flooding caused by climate changes leads to oxygen deficiency of the soil (hypoxia), which reduces the photosynthetic rate, disrupts photosynthate transport, and decreases ATP production in roots (Visser et al., 2015; Birnbaum et al., 2017). Furthermore, flooding adversely affects nutrient uptake and transport in plants, leading to growth reduction of plants. Functional morphological and anatomical features of plants can adapt to flooding conditions, such as hypertrophied lenticels and adventitious roots (water roots) (Birnbaum et al., 2017). Although Cu and Zn at low levels are essential for plant growth and involved in numerous physiological processes; however, at high concentrations, they are actively toxic to plant metabolism such as photosynthesis and respiration (MacFarlane et al., 2007; Marques et al., 2007; Padmavathiamma and Li, 2010). Therefore, for poorly drained soils, plants have to cope with combined stresses of heavy metals and flooding. Heavy metals and their interaction with flooding become the major abiotic stress factors which compromise plant growth and phytoremediation potentials. Further, the alteration between oxic and anoxic conditions will influence phytoremediation modes such as phytoextraction or phytostabilization (Poot et al., 2007). Phytoremediation plants should adopt the shifts from environmental conditions caused by climate changes. Climate changerelated factors influence phytoremediation potentials such as elevated CO2 (Wang et al., 2012), increasing temperature (Li et al., 2012), flooding (Ferreira et al., 2019), drought (Han et al., 2013), and salinization (Van Oosten and Maggio, 2015). Therefore, similar to climate-smart agriculture (Valliyodan et al., 2016), it should be proposed climate-smart phytoremediation to address the challenges from environmental degradation and climate changes.

Meanwhile, metal speciation and phytoavailability in the rhizosphere are very important for phytoremediation application (Du Laing et al., 2009; Domínguez et al., 2011), and removing or restricting phytoavailable heavy metals in the rhizosphere determines selection between phytoextraction and phytostabilization (Almeida et al., 2009; Padmavathiamma and Li, 2010). Plants can modify metal phytoavailability at the substrates surrounding the  et al., 2010). Therefore, the management roots (Martínez-Alcala measures for reducing metal bioavailability and retaining metals in the rhizosphere decrease environmental risks (Du Laing et al., 2009; Domínguez et al., 2011). Changes of heavy metals in the rhizosphere can be assessed through single chemical extraction procedures because of their simplicity, low cost and smooth operation such as chelate EDTA and neutral salts (e.g., CaCl2 and NH4NO3) (Song et al., 2004; Soriano-Disla et al., 2010a). The willow (Salix spp.) is an excellent candidate for phytoremediation of soil owing to high tolerance to heavy metals, large biomass production, extensive root systems, and wide adaption to different environmental conditions such as flooding, salinity and drought (Watson et al., 2003; Kuzovkina and Volk, 2009). Furthermore, short rotation coppice (SRC) based on Salix clones provides opportunities for biomass energy production and carbon sequestration; therefore, phytoremediation in terms of willows is considered a suitable method to decrease environmental risks of pollutants and mitigate climate changes (Jensen et al., 2009; Yang et al., 2014). Plants respond to heavy metals and flooding depending on species or clones; Salix spp. exhibits broad genetic diversity in accumulating metal capacity and adoption to multiple environmental stresses, which provides a precious resource to select superior Salix clones to increase phytoremediation capacity in flooded sites (Kuzovkina and Volk, 2009; Yang et al., 2014). This study mainly determined the effects of flooding on phytoremediation capacity of Cu and Zn by willows. Therefore, the aims of this study were to (1) compare uptake and accumulation of Cu and Zn across Salix clones under flooded versus non-flooded conditions; (2) examine the changes of Cu and Zn in the rhizosphere across the Salix clones under flooded versus non-flooded conditions using simple extraction procedures. The obtained results will provide new insight into phytomanagement of flooded contaminated sites by willows. 2. Materials and methods 2.1. Plant materials The six excellent shrub willow clones used in this study were obtained from China National Willow Germplasm Resource in Jiangsu Academy of Forestry, Nanjing, China, i.e., clones J1052 (Salix suchowensis  S. leucopithecia), SS61 (S. suchowensis), SI63 (S. integra), SV683 (S. viminalis), JW8-26 (S. suchowensis  S. intergra), and JW9-6 (S. intergra  S. suchowensis). These clones have excellent growth performance, which have been widely planted in wet environments in southeast China. The cuttings (15 cm long) of the clones were directly inserted into the rubber insulation sheets (2 cm thickness), then floated in the tanks with 1/4 strength Hoagland nutrient solution for rooting and pre-growing for 30 d (Yang et al., 2014). The rooted plantlets

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with relatively uniform size were selected for this experiment. 2.2. Soil collection In this study, the sampling site is located in a typical Cu- and Zncontaminated area in Hangzhou (29110 e30 340 N; 118 200 e120 370 E), China, and metal recycling industries had contributed to the enhancement of Cu and Zn contents in the soil. This region belongs to the humid subtropical climate, and heavy precipitation often occurs from July to September, causing seasonal flooding. The soil was collected from the plow layer (0e30 cm), air-dried, gently ground to sieve (1-cm mesh size) and homogenized, and the main physical-chemical parameters of the soil were analyzed based on the Chinese standardized method. The soil properties were as follows: pH 6.2 ± 0.1, silt (%) 17.0 ± 1.8, clay (%) 60.0 ± 3.3, sand (%) 14.0 ± 3.2, organic matter (g kg1) 15.8 ± 0.6, CEC (cmol kg1) 11.4 ± 1.3, available N (mg g1) 78.6 ± 1.1, available P (mg g1) 0.30 ± 0.02, available K (mg g1) 270.3 ± 2.0. Total Cu and Zn concentrations in the soil are 2414.5 ± 67.3 mg g1 and Zn 4623.2 ± 89.6 mg g1, respectively, which far exceed the limits (400 mg g1 Cu and 500 mg g1 Zn) of the third grade of the Chinese environmental quality standard for soils (GB15618-1995). 2.3. Experiment setup A pot experiment was carried out in a greenhouse (manly sheltering from rainfall) from July to October in 2017 at Zhejiang University, Hangzhou, China. To avoid nutrient deficiencies, the soil substrate was pre-mixed with fertilizer containing 60 mg kg1 nitrogen (N) as urea, 80 mg kg1 phosphorus (P) as KH2PO4, and 70 mg kg1 potassium (K) as K2SO4, and as a solution, these nutrients were added to the soil and mixed thoroughly before the experiment. The whole experiment was a completely randomized factorial design with two factors and three replicates, i.e., clones and treatments (non-flooded (control) and flooded treatments). Four 30-day seedlings of each Salix clone were transplanted to a pot (four plants per pot) with 10 kg soil. After potted seedlings acclimated to grow under the current condition for a week, they were subjected to the treatments. At control group (non-flooded treatment), the potted Salix clones were maintained 70% water holding capacity by periodic addition of deionized water; at treatment group, the potted Salix clones received flooded treatments with 5 cm of water on the top of the soil surface during the whole experiment. The pots were randomly placed in the greenhouse; except rainfall, the main conditions such as temperature, humid and natural photoperiod remained consistent with the current field conditions. 2.4. Single extraction procedure After the experiment of 100 d, flooded pots were drained for a day, and soil samples and rhizospheric soils (the rhizospheric soil was defined as the soil which was surrounded by dense roots (Greger and Landberg, 2008)) across the Salix clones were collected under both non-flooded and flooded treatments. Extractable Cu and Zn in the rhizosphere across the Salix clones were assessed following three single extraction procedures (0.05 M EDTA, 1 M NH4NO3 and 0.01 M CaCl2) (Song et al., 2004; Meers et al., 2007; Soriano-Disla et al., 2010a, 2010b). 2.5. Chemical analyses At the end of the experiment, plants were harvested and washed

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with deionized water; further, plant samples were divided into leaves, stems and roots, dried at 65  C and weighed for plant biomass. The plant material was grinded and digested with a mixture of HNO3: HClO4 (4:1 v/v) in a hot block system (ED36, LabTech, Germany). The soil samples were air-dried, homogenized, ground to sieve (<2 mm), and digested with HNO3þHClO4þHF in closed Teflon vessels using the microwave oven (PerkinElmer, Multiwave) at 180  C. The Cu and Zn concentrations in plants, soil samples and extractants were determined by an inductively coupledeplasma mass spectrometer (ICP-MS; Agilent 7500a, Agilent Technologies, Santa Clara, CA, USA). Macronutrient N, P and K in plant tissues were also measured based on the Chinese standardized method (the result see Supplementary Table 1). The certified reference samples from China (the brown soil and the tomato leaves) were analyzed through metal determination analytical method. 2.6. Calculations and statistical analysis Translocation factor (TF) was calculated as the ratio of metal concentrations in leaves to roots, and bioconcentration factor (BCF) for each metal was determined as the ratio of metal concentrations in leaves to those of the soil. The phytoextraction potential (shoot metal content) was calculated based on shoot biomass (including leaf and stem): shoot metal content ¼ leaf biomass  leaf metal concentration þ stem biomass  stem metal concentration; the phytostabilization potential mainly depended on root metal content: root metal content ¼ root biomass  metal concentration in roots. The data were analyzed using two-way analysis of variance (ANOVA) followed by Tukey post-hoc test (P < 0.05) for the effects of clone, treatment and their interaction. When the parameters showed significance after the analysis of two-way ANOVA, they were further analyzed using one-way ANOVA with Turkey post-hoc test (P < 0.05) or Student’s test (P < 0.05) (see Supplementary Tables 2 and 3). The statistical analysis was carried out using SPSS 16.0 (SPSS Inc., Chicago, IL, USA); the data presented were means ± SE of three replicates (n ¼ 3). 3. Results 3.1. Plant growth Under well-drained (control) conditions, all Salix clones grew well without showing toxicity systems (such as leaf chlorosis and nutrient deficiency) and no occurrence of adventitious roots. In contrast, upon flooding, visible adventitious root primordia occurred; leaves exhibited slight chlorosis; the leaf areas and seedling height decreased; the adventitious roots and hypertrophied lenticels developed on submerged portions of stems. However, all Salix clones still survived during 100-day flooded stress. Fig. 1 shows the biomass accumulation of Salix clones under non-flooded and flooded conditions. The only treatment factor significantly affected leaf biomass across the Salix clones (Table 1, P < 0.01). The leaf biomass varied by 2.5-fold (non-flooded) and 2.4fold (flooded) among the clones; the leaf biomass of flooded Salix clones decreased by 26% (J1052), 71% (SS61), 81% (SI63), 57% (SV683), 81% (JW8-26), and 28% (JW9-6) compared to the corresponding non-flooded clones (Fig. 1a). The only treatment factor significantly influenced stem biomass across the clones (Table 1, P < 0.01). The stem biomass differed by 3.6-fold (non-flooded) and 2.5-fold (flooded) across the clones (Fig. 1b); the flooded clones reduced stem biomass by 40% (J1052), 67% (SS61), 45% (SI63), 70% (SV683), 67% (JW8-26), and 17% (JW96) compared to the respective non-flooded clones.

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Fig. 1. Biomass accumulation of the Salix clones grown under flooded versus non-flooded conditions for 100 d. Values present mean ± SE (n ¼ 3).

Table 1 Summary of two-way ANOVA for various parameters affected by clones and flooding. Clone  treatment

Source

Clone

Treatment

F-value

P-value

F-value

P-value

F-value

P-value

Leaf biomass Stem biomass Root biomass Leaf Cu concentration Stem Cu concentration Root Cu concentration Leaf Cu TF Leaf Cu BCF Leaf Zn concentration Stem Zn concentration Root Zn concentration Leaf Zn TF Leaf Zn BCF EDTA-Cu CaCl2eCu NH4NO3eCu EDTA-Zn CaCl2eZn NH4NO3eZn Shoot Cu content Root Cu content Shoot Zn content Root Zn content

0.377 2.529 5.747 1.880 6.682 3.358 11.700 0.926 11.419 4.507 1.772 3.658 8.136 2.561 2.371 0.658 2.527 0.531 0.790 0.944 11.175 1.541 5.355

0.860 0.056 0.001 0.135 0.000 0.019 0.000 0.481 0.000 0.005 0.157 0.013 0.000 0.054 0.070 0.309 0.057 0.751 0.567 0.471 0.000 0.215 0.002

10.124 22.134 0.635 31.858 0.095 101.589 102.507 0.359 118.497 34.413 25.374 74.799 102.503 0.754 9.130 30.222 0.341 60.253 2.321 31.591 28.008 27.935 6.750

0.004 0.000 0.433 0.000 0.760 0.000 0.000 0.555 0.000 0.000 0.000 0.000 0.000 0.394 0.006 0.000 0.565 0.000 0.141 0.000 0.000 0.000 0.016

0.673 1.873 5.009 1.657 1.235 1.896 11.478 1.106 4.059 1.926 2.648 2.063 5.689 1.577 0.658 1.521 1.840 4.381 0.804 0.898 11.105 1.256 4.503

0.648 0.137 0.003 0.184 0.324 0.133 0.000 0.383 0.008 0.127 0.048 0.106 0.001 0.204 0.658 0.221 0.143 0.006 0.558 0.499 0.000 0.315 0.005

Note: Significant effects are highlighted by P-values in Bold (P < 0.05).

The clone factor and the clone  treatment significantly impacted root biomass across the Salix clones (Table 1, P < 0.01). Different Salix clones significantly differed in root biomass, varying by 2.1-fold (non-flooded) and 1.9-fold (flooded); flooded treatments increased the root biomass of J1052 (35%), JW8-26 (52%) and JW9-6 (65%), and decreased SS61 (69%), SI63 (78%) and SV683 (64%)

compared to the non-flooding treatments (Fig. 1c). 3.2. Cu uptake accumulation upon flooding versus non-flooding Fig. 2 shows tissue Cu concentrations, leaf Cu BCFs and Cu TFs of Salix clones under two conditions. Overall, distribution patterns of

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Fig. 2. Tissue Cu concentrations, leaf Cu TFs and leaf Cu BCFs of the Salix clones grown under flooded versus non-flooded conditions for 100 d. Values present mean ± SE (n ¼ 3).

Cu across the clones decreased in the following order: root > leaf > stem under two treatments. Only treatment factor had a significant effect on leaf concentrations across the clones (Table 1, P < 0.01). The clonal differences in leaf Cu concentrations ranged from 90.1 mg g1 (SV683) to 447.2 mg g1 (SI63) under non-flooded condition, and from 18.3 mg g1 (SS61) to 35.6 mg g1 (SI63) under flooded condition (Fig. 2a). Flooded clones reduced leaf Cu concentrations by 94% (J1052), 90% (SS61), 92% (SI63), 75% (SV683), 92% (JW8-26), and 86% (JW9-6) compared to the respective nonflooded clones. Only clone factor significantly influenced stem Cu concentrations (Table 1, P < 0.01). Stem Cu concentrations across the Salix clones varied from 27.8 mg g1 (JW8-26) to 51.4 mg g1 (SI63) under non-flooded condition, and from 23.4 mg g1 (JW9-6) to 69.8 mg g1 (SI63) under flooded condition; stem Cu concentrations across the clones were small variation between non-flooded and flooded conditions (Fig. 2b). Every factor (clone or treatment) had significant effects on root Cu concentrations (Table 1, P < 0.05). Root Cu concentrations

among the clones ranged from 1120.0 mg g1 (SS61) to 1792.4 mg g1 (SI63) under non-flooded condition, and from 2539.4 mg g1 (SV683) to 4419.5 mg g1 (JW8-26) under flooded condition; flooded clones increased root Cu concentrations, by 1.4- (J1052), 1.9(SS61), 1.2- (SS63), 1.2- (SV683), 2.8- (JW8-26), and 1.3-fold (JW9-6) compared to the non-flooded clones. Two factors and their interaction had significant effects on leaf Cu TFs (Table 1, P < 0.01). Overall, leaf Cu TFs were relatively low under two conditions; flooded clones decreased leaf Cu TFs compared to respective non-flooded clones (Fig. 2d). Leaf Cu BCFs were less variation among the clones and between treatments and had relatively low values under both conditions (Fig. 2e). 3.3. Zn uptake and accumulation upon flooding versus nonflooding Fig. 3 presents tissue Zn concentrations, leaf Zn TFs and leaf Zn BCFs of Salix clones under two treatments. Every factor and their interaction had significant effects on leaf Zn concentrations

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Fig. 3. Tissue Zn concentrations, leaf Zn TFs and leaf Zn BCFs of the Salix clones grown under flooded versus non-flooded conditions for 100 d. Values present mean ± SE (n ¼ 3).

(Table 1, P < 0.01). Leaf Zn concentrations across the clones varied between 761.4 mg g1 (JW9-6) and 2195.5 mg g1 (SV683) under non-flooded condition, and between 204.9 mg g1 (J1052) and 636.7 mg g1 (SV683) under flooded condition; flooded clones dramatically decreased leaf Zn concentrations by 78% for J1052, 73% for SS61, 78% for SI63, 71% for SV683, 69% for JW8-26, and 53% for JW9-6 compared to the corresponding non-flooded clones (Fig. 3a). Two factors had significant effects on stem Zn concentrations (Table 1, P < 0.01). Stem Zn concentrations among the clones differed from 123.7 mg g1 for JW8-26 to 184.1 mg g1 for SI63 under non-flooded condition, and from 88.0 mg g1 for J1052 to 139.5 mg g1 for SV683 under flooded condition, and flooded clones lowered stem Zn concentrations by 42% for J1052, 42% for SS61, 26% for SI63, 15% for SV683, 16% for JW8-26, and 14% for JW9-6 compared to the non-flooded clones (Fig. 3b). The treatment factor and the clone  treatment had significant impacts on root Zn concentrations (Table 1, P < 0.05). Zn mainly accumulated in roots under both conditions, especially under flooded condition. Root Zn concentrations across the clones varied

between 1036.0 mg g1 (J1052) and 1543.2 mg g1 (JW9-6) under non-flooded condition, and between 1497.1 mg g1 (JW9-6) and 2773.3 mg g1 (SS61) under flooded condition. Except for JW9-6 (decreased by 3% compared to non-flooded treatment), flooded clones increased root Zn concentrations by 100% (J1052), 130% (SS61), 56% (SI63), 17% (SV683), and 97% (JW8-26) compared to the corresponding non-flooded clones. Two factors showed significant effects on leaf Zn TFs (Table 1, P < 0.05). The ranges of Leaf Zn TFs were from 0.50 to 1.58 (nonflooded), and from 0.10 to 0.25 (flooded); flooded clones dramatically decreased their leaf Zn TFs compared to the non-flooded state (Fig. 3d). Every factor and their interaction significantly affected leaf Zn BCFs (Table 1, P < 0.01); flooded clones decreased their leaf Zn BCFs compared to those of non-flooded treatments (Fig. 3e). 3.4. Changes of Cu and Zn in the rhizosphere Fig. 4 presents extractable Cu and Zn by EDTA, NH4NO3 and CaCl2 in the rhizosphere across the clones under two conditions.

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Fig. 4. The changes of extractable Cu and Zn in the rhizosphere of the Salix clones grown under flooded versus non-flooded conditions for 100 d. Values present mean ± SE (n ¼ 3).

Extractable Cu across the clones decreased in the order: EDTA > NH4NO3>CaCl2. Two factors and their interaction showed no significant effects on EDTA-extractable Cu (Table 1). EDTA-extractable Cu had slight variation among the clones under two conditions; EDTAextractable Cu of J1052, SS61 and SI63 under flooded treatments was higher than that under non-flooded conditions; conversely, EDTA-extractable Cu of SV683, JW8-26 and JW9-6 under flooded condition was lower than under non-flooded conditions (Fig. 4a). Only treatment factor significantly affected CaCl2-extractable Cu across the clones (Table 1, P < 0.01). Different Salix clones had marked differences in CaCl2-extractable Cu, and amounts of CaCl2extractable Cu of flooded clones were higher than those of corresponding non-flooded clones (Fig. 4b). The treatment factor had a significant effect on NH4NO3extractable Cu across the clones (Table 1, P < 0.01). There was small

clonal variation in NH4NO3-extractable Cu under two treatments, and quantities of NH4NO3-extractable Cu of flooded Salix clones were higher than those of non-flooded clones (Fig. 4c). As shown in Fig. 4, extractable Zn across the clones decreased in the order: EDTA > NH4NO3>CaCl2. The two factors and their interaction had no significant effects on EDTA-extractable Zn. Different Salix clones had slight variation in EDTA-extractable Zn under both conditions, and flooded treatments had fewer impacts on EDTAextractable Zn across the clones compared to the control (Fig. 4d). The treatment factor and the clone  treatment had significant effects on CaCl2-extractable Zn (Table 1, P < 0.01). Except for clone J1052, amounts of CaCl2-extractable Zn of the flooded Salix clones were lower than those of non-flooded clones (Fig. 4e). Two factors and their interaction did not show significant effects on NH4NO3-extractable Zn (Table 1). Flooded treatments increased NH4NO3-extractable Zn of J1052, SS61 and JW9-6, while slightly

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decreased NH4NO3-extractable Zn of SI63, SV683 and J8-26 compared to the identical clones of the non-flooded treatments (Fig. 4f). 3.5. Phytoremediation potentials upon flooding versus non-flooding Fig. 5 shows shoot Cu/Zn contents and root Cu/Zn contents under two conditions. Only treatment had a significant impact on shoot Cu contents among the clones (Table 1; P < 0.01). The ranges of shoot Cu contents were from 440 mg plant1 (JW9-6) to 1166 mg plant1 (JW8-26), varying by 2.6-fold (non-flooded), and from 99 mg plant1 (SV683) to 130 mg plant1 (SI63), changing by 1.3-fold (flooded) among the clones; flooded clones decreased shoot Cu contents by 6.2- (J1052), 10- (SS61), 6.8- (SI63), 5.1- (SV683), 9.3(JW8-26), and 3.7-fold (JW9-6) compared with the corresponding non-flooded clones (Fig. 5a). Two factors and their interaction had significant effects on root Cu contents (Table, P < 0.01). Different Salix clones showed 2-fold (non-flooded) and 21-fold (flooded) variation in root Cu contents, ranging from 873 mg plant1 (JW9-6) to 1726 mg plant1 (SS61) under non-flooded condition, and from 575 (SI63) and 12,196 mg plant1 (JW8-26) under flooded condition. Flooded treatments increased root Cu contents by 4-fold for J1052, 8.2-fold for JW8-26 and 6.4-fold for JW9-6, whereas reduced by 1.2-fold for SS61, 2.0fold for SI63 and 1.2-fold for SV683 compared with the identical clones of non-flooded treatments (Fig. 5b). Only treatment factor significantly affected shoot Zn contents across the clones (Table 1, P < 0.01). Different Salix clones had 3.2fold (non-flooded) and 3.0-fold (flooded) variation in shoot Zn contents, varying from 2051 mg plant1 (JW9-6) to 6754 mg plant1 (SV683) for control, and from 358 mg plant1 (SI63) to 1102 mg

plant1 (SV683) for treatments. The flooded clones decreased the shoot Zn contents by 4.5-fold for J1052, 9.0-fold for SS61, 9.3-fold for SI63, 6.1-fold for SV683, 9.0-fold for JW8-26, and 2.1-fold for JW9-6 compared with the non-flooded clones (Fig. 5c). Every factor and their interaction significantly influenced root Zn contents (Table 1, P < 0.05). Root Zn contents across the clones varied from 795 mg plant1 for SI63 to 1927 mg plant1 for SS61 (non-flooded), and from 491 mg plant1 for JW8-26 to 1102 mg plant1 for SV683 (flooded); flooded treatments reduced root Zn contents by 1.3-fold for SS61, 3.0-fold for SI63 and 2.3-fold for SV683, while increased 3.3-fold for J1052, 4.5-fold for JW8-26 and 2.7-fold for JW9-6 compared with non-flooded treatments (Fig. 5d). 4. Discussion 4.1. Plant morphology and growth Flooding reduces the diffusion of oxygen into the soil, leading to soil anoxia and oxygen deficiency for roots (Birnbaum et al., 2017). In general, willows can grow under the hypoxic condition and adapt to frequent and or prolonged flooding (Kuzovkina and Volk, 2009). Soils are heterogeneous and soil prosperities vary from one place to another; it was difficult to find the ideal soil control as compared to the tested soil; therefore, in this study, it was mainly considered how flooding stress affected the Salix clones. All tested non-flooded Salix clones showed an extremely high tolerance to the current contaminated soil. In contrast, decreases in leaf areas and chlorosis of the flooded clones showed that flooding markedly disrupted their photosynthesis. Furthermore, all Salix clones could survive under relatively long-term flooding stress, which was attributed to the rapid development of lenticels and aquatic

Fig. 5. Shoot Cu/Zn contents and root Cu/Zn contents of the Salix clones grown under flooded versus non-flooded conditions for 100 d. Values present mean ± SE (n ¼ 3).

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adventitious roots (Li et al., 2006; Nakai et al., 2009). The formation of lenticels and adventitious roots meets oxygen demands for roots; and meanwhile adventitious roots restore root functioning to take up nutrients dissolved in the floodwater (Birnbaum et al., 2017). The clones J1052, JW8-26 and JW9-6 had superior capacities to produce adventitious roots, especially for JW9-6, conferring their tolerance to both heavy metals and flooding. All flooded Salix clones showed sharp reductions in leaf and stem biomass production, varying with clones, especially for leaves, suggesting that leaves were sensitive to flooding stress, such as clones JW8-26 and SS61. Reduction in the root biomass of SS61, SI63 and SV683 suggested that three Salix clones were vulnerable to flooding despite the formation of adventitious roots. Conversely, flooding pronouncedly enhanced the root biomass accumulation of J1052, JW8-26 and JW9-6; this can be explained that the outgrowth of aquatic adventitious roots contributed to the total root dry mass of these clones. Flooding lowered shoot: root ratios across the Salix clones, indicating that the Salix clones allocated higher biomass to the root system under flooding condition (Possen et al., 2011). This result is consistent with the reports concerning several willow species, such as S. nigra (Li et al., 2006) and S. gracilistyla (Nakai et al., 2009). Under the non-flooding condition, the best growth performance was SI61; under flooding condition, JW9-6 had the best growth performance.

4.2. Uptake and accumulation of Cu and Zn Phytoremediation efficiency depends on several factors, such as plant species, soil properties, metal bioavailability, and environmental conditions (Vandecasteele et al., 2005; Jensen et al., 2009). The result showed that different Salix clones had wide variation in uptake and accumulation of Cu under two treatments. The welldrained Salix clones exhibited relatively high Cu accumulation in aerial tissues, especially in leaves, such as J1052, SI63 and JW8-26, although large amounts of Cu accumulated in roots. It can be explained that under aerobic environment, the Salix clones had better growth performances and produced relatively large biomass to facilitate Cu extraction from the soil; however, both Cu BCFs and TFs had relatively low values (<1), confirming that Cu had relatively weak translocation capacity within plants (Kopponen et al., 2001; MacFarlane et al., 2007; Zimmer et al., 2011). In contrast to nonflooded Salix clones, leaf Cu concentrations of the flooded Salix clones dramatically decreased despite little impacts on stem Cu concentrations; the most significant amounts of Cu retained in roots with extremely low Cu TFs and Cu BCFs. Therefore, it was concluded that the Salix clones were Cu excluders under flooding condition, such as JW8-26 and JW9-6. Similarly, different Salix clones showed wide differences in uptake and accumulation of Zn under two treatments. The nonflooded Salix clones accumulated large amounts of Zn in aerial parts with high leaf Zn TFs (values of SI63 and SV683 were >1), showing that the Salix clones had good ability to transport Zn from roots to aerial parts. Contrary to non-flooding, flooding largely decreased aerial part Zn concentrations across the Salix clones with low leaf Zn TFs, and the largest amounts of Zn accumulated in roots, suggesting that flooding considerably limited Zn transportation from roots to shoots. Similar to the result, Vandecasteele et al. (2005) showed that longer submersion periods in the field lowered leaf Zn concentrations in the willows. The well-drained Salix clones displayed accumulation capacities of Cu and Zn in the aerial parts, although strongly depending on Salix clones and metal types. Conversely, the flooding system significantly decreased the translocation and accumulation of Cu/ Zn in aerial parts, resulting in large amounts of Cn/Zn stored in

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 et al., 2008). After subjected to flooding, roots over time (Cambrolle growth inhibition and reduction of both biomass production and metal concentrations were primary factors for decreasing Cu/Zn accumulation in aerial tissues, which may be attributed to the following causes. Flooding causes oxygen deficiency and dramatically inhibits photosynthesis and shoot biomass production (Li et al., 2006; Nakai et al., 2009). The uptake of heavy metals is transpiration-mediate transport and closely related to transpiration rate (Rennenberg et al., 2009; Santana et al., 2012); only small amounts of metals are transported from roots to shoots due to reduction of stomatal conductance and low transpiration caused by flooding stress, particular for Cu (Salah and Barrington, 2006; Birnbaum et al., 2017). The heavy metal transport is energyconsuming processes, and the switch from aerobic to anaerobic metabolism yields the low amount of ATP in roots via oxidative phosphorylation to reduce uptake and transportation of heavy metals (Rennenberg et al., 2009; Kreuzwieser and Gessler, 2010; Visser et al., 2015; Striker and Colmer, 2016). Flooding reduces the availability of transporter proteins and represses transportation capacity of transporters to decrease active transport processes of heavy metals (Kreuzwieser and Gessler, 2010). Meanwhile, the present result also showed that Cu and Zn had different mobility within the Salix clones. Although both Cu and Zn are essential elements for plants, the ratios of leaf Zn/Cu concentration (2.4e24 for non-flooding and 6.8e28 for flooding across the Salix clones) reflected that the Salix clones preferentially took up more Zn than Cu, indicating that Zn was more mobile and phytoextractable than Cu (Fuentes et al., 2007; MacFarlane et al., 2007; Zimmer et al., 2011). Similarly, Jeyakumar et al. (2010) reported that Cu accumulation was lower in Poplar leaves than Zn. Except clones J1052 and SI63, the other non-flooded clones showed higher uptake efficacy of Zn in roots compared to Cu, whereas flooded clones accumulated and stored more Cu in roots than Zn, or Cu had poorer transportation capacity than Zn (Kopponen et al., 2001; Fuentes et al., 2007; MacFarlane et al., 2007; Jeyakumar et al., 2010). The result also revealed that flooding had larger effects on Cu than Zn (Kopponen et al., 2001).

4.3. Changes of Cu and Zn in the rhizosphere Phytoremediation abilities of heavy metals are associated with extractable metal (phytoavailable metals) rather than total metal concentrations in the soil (Domínguez et al., 2011; Zimmer et al., 2011). Plants and environmental factors impact the speciation  et al., 2008; Almeida et al., and bioavailability of metals (Cambrolle 2009; Huang et al., 2011). In this study, the quantities of EDTA-extractable Cu/Zn were closest to and correlated with Cu concentrations in roots and Zn concentrations in leaves and roots across the Salix clones (see Supplementary Table 4), confirming that root Cu concentration is a good indicator of Cu bioavailability (Bravin et al., 2010). Similarly, Brun et al. (2001) reported that root Cu concentration was correlated with EDTA-extractable Cu. On the other hand, the result revealed that EDTA extractant was suitability for the soil type and the plant species. EDTA solubilize metals in absorbed and organically bound phases and is suitable for the acid soil (Soriano-Disla et al., 2010a, 2010b). However, extractable yields of Cu/Zn by CaCl2 and NH4NO3 were far lower compared to Cu/Zn concentrations in leaves and roots and had poor relationship with tissue Cu/Zn concentrations, especially for CaCl2-extractable Cu, suggesting that CaCl2 and NH4NO3 were not suitable to evaluate phytoavailable Cu/Zn to the Salix clones under the current condition. This might be explained that the clay was accounted for a high proportion in the soil, and CaCl2 and

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W. Yang et al. / Chemosphere 241 (2020) 125059

NH4NO3 are not suitable to extract metals in the upper clay soil, whereas they are appropriate for the heavy metals mostly located on mineral surfaces and displaced by other cations (Soriano-Disla et al., 2010a, 2010b). The current result differed from the report by Song et al. (2004), who showed that NH4NO3 extraction was suitable to assess phytoavailability of Cu in the contaminated soils, while EDTA extraction may overestimate plant available Cu. Plant roots can increase mobility and change the speciation of heavy metals through excreting organic acids. Therefore, comparisons among different extraction procedures were complex to estimate metal bioavailability owing to different soil properties and plant species (Song et al., 2004; Meers et al., 2007; Soriano-Disla et al., 2010a, 2010b). Quantities of extractable Zn by CaCl2 and NH4NO3 were higher than those of extractable Cu in the rhizosphere by CaCl2 and NH4NO3, confirming that Zn was more easily extracted than Cu (Jeyakumar et al., 2010); this is probably because Cu forms strong complexes with organic matter (Song et al., 2004; Soriano-Disla et al., 2010a, 2010b). Different Salix clones showed different mobilization ability for Cu/Zn in their rhizosphere soil (Domínguez et al., 2011). Hydrological factors also influenced extractable Cu and Zn in the rhizosphere, depending on clones and metal types, and flooding resulted in larger increases in CaCl2-extractable and NH4NO3-extractable Cu as compared to the control, which is be explained that the flooded clones developed more adventitious roots to mobilize metals in the rhizosphere. The result differed from the report that flooded soil caused the transformation of Cu into less soluble forms (Huang et al., 2011). Except J1052, flooding decreased NH4NO3-extractable Zn compared to the control. Similarly, Vandecasteele et al. (2005) reported that submerged soil had the lowest Zn availability; Wang et al. (2009) showed that soil moisture influenced the distribution of Zn in the rhizosphere of maize. 4.4. Phytoremediation potentials upon flooding versus non-flooding Phytoremediation capacity was strongly associated with both tissue metal accumulation and biomass production (Jensen et al., 2009; Yang et al., 2014). It is essential for phytoremediation application to examine the allocation of heavy metals between root (belowground) and shoot (aboveground) levels to determine phytoextraction or phytostabilization (Marques et al., 2007; Padmavathiamma and Li, 2010). Under non-flooded conditions, the ratios of shoot Cu content/root Cu content (0.36e0.79 across the Salix clones) revealed that non-flooded clones had some phytoextraction potentials of Cu; high ratios of shoot Zn content/root Zn content (1.7e4.1) exhibited that the Salix clones had better Zn phytoextraction capacities. This result also confirmed that Zn was more phytoextractable than Cu (MacFarlane et al., 2007; Zimmer et al., 2011). Therefore, the aerobic condition provides a favorable environment benefiting plant growth and increasing phytoextraction ability despite heavy Cu and Zn contamination. However, under flooding condition, low ratios of shoot Cu content/root Cu content (0.01e0.22) displayed sparse phytoextraction potentials of Cu (in fact, showing phytostabilization traits for Cu). The ratios of shoot Zn content/root Zn content (0.28e0.81) indicated that flooding markedly decreasing phytoextraction capacity; however, some clones still performed relatively high phytoextraction potential such as SV683. The dramatic decrease in Cu/ Zn shoot contents (i.e., phytoextraction potentials) was attributed to a substantial reduction of both shoot biomass and aerial tissue metal concentrations. Interestingly, flooding increased the root biomass of some clones owing to superior adventitious rooting capacities such as JW8-26 and JW9-6; meanwhile, flooding also limited metal transportation from roots to shoots, resulting in most

of the metals stored in roots. Therefore, the flooded Salix clones exhibited better phytostabilization rather than phytoextraction. Consequently, phytostabilization is considered as one of the most effective alternatives to remediate flooded contaminated soils by willows (Padmavathiamma and Li, 2010). Based on the present results, it is predicted that when flooding occurs, phytoextraction function of willows shifts to phytostabilization; when flooded soil is drained, the willows will restore phytoextraction function. Therefore, regardless of phytoextraction or phytostabilization, it is concluded that the willows still play a decisive role in phytoremediation when an extreme flooding event occurs. Under non-flooded and flooded conditions, SV683 showed the largest co-phytoextraction potential of Cu and Zn; under the flooded condition, JW8-26 and JW9-6 showed better cophytostabilization potentials of Cu and Zn. Therefore, another important implication is that selecting flooding-tolerant and high accumulating metal Salix clones improves phytoremediation potentials under a wet environment. 5. Conclusions The tested Salix clones grew well under the well-drained condition; although flooding strongly inhibited shoot growth of Salix clones to different extents, they developed hypertrophied lenticels and adventitious roots, conferring 100% survival under combined stresses of heavy metals and flooding. Different Salix clones showed wide variation in biomass production and accumulation of Cu and Zn under two treatments. The non-flooded Salix clones could accumulate more Cu and Zn in aerial parts coupled with higher aboveground biomass, especially for Zn, showing phytoextraction capacities of Cd/Zn despite clonal differences. In contrast, the flooded clones caused more considerable reductions in accumulation of Cu and Zn in aerial parts, markedly decreasing phytoextraction capacities of Cu and Zn; the most massive amounts of Cu and Zn were retained in roots combined with superior root biomass, exhibiting promising phytostabilization potentials of Cu/Zn, especially for Cu. EDTA-extractable Cu/Zn in the rhizosphere was suitable to predict phytoavailability of Cu and Zn to the Salix clones under the current condition. Irrespective of phytoextraction (non-flooding) or phytostabilization (flooding), the effects of heavy metals on the environment should be minimized through phytoremediation with willows. Another implication is that the selection of Salix clones for phytoremediation applications should focus on not only metal concentrations and biomass production but also wide adaption to environmental changes. Acknowledgments This work was funded by the National Natural Science Foundation of China (31100513), Major Science and Technology Program of Hainan Province (ZDKJ2017002) and China Scholarship Council (201708460027). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2019.125059. References Almeida, C.M.R., Dias, A.C., Mucha, A.P., Bordalo, A.A., Vasconcelos, M., 2009. Influence of surfactants on the Cu phytoremediation potential of a salt marsh plant. Chemosphere 75, 135e140. https://doi.org/10.1016/ j.chemosphere.2008.12.037. Birnbaum, D., de Kroon, H., Huber, H., Zhang, Q., de Best, S., Beljaars, S.J.M.,

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