Phosphorus influence Cd phytoextraction in Populus stems via modulating xylem development, cell wall Cd storage and antioxidant defense

Journal Pre-proof Phosphorus influence Cd phytoextraction in Populus stems via modulating xylem development, cell wall Cd storage and antioxidant defense Can Yang, Wenwen Qiu, Zexin Chen, Wenyi Chen, Yunfei Li, Jingle Zhu, Siddiq Ur Rahman, Zixuan Han, Yun Jiang, Guijuan Yang, Jing Tian, Qin Ma, Yi Zhang PII:

S0045-6535(19)32393-8

DOI:

https://doi.org/10.1016/j.chemosphere.2019.125154

Reference:

CHEM 125154

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ECSN

Received Date: 19 August 2019 Revised Date:

12 October 2019

Accepted Date: 19 October 2019

Please cite this article as: Yang, C., Qiu, W., Chen, Z., Chen, W., Li, Y., Zhu, J., Rahman, S.U., Han, Z., Jiang, Y., Yang, G., Tian, J., Ma, Q., Zhang, Y., Phosphorus influence Cd phytoextraction in Populus stems via modulating xylem development, cell wall Cd storage and antioxidant defense, Chemosphere (2019), doi: https://doi.org/10.1016/j.chemosphere.2019.125154. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

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Phosphorus influence Cd phytoextraction in Populus stems via modulating

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xylem development, cell wall Cd storage and antioxidant defense

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Can Yanga, 1, Wenwen Qiua, 1, Zexin Chena, Wenyi Chena, Yunfei Lia, Jingle Zhub, Siddiq

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Ur Rahmanc, Zixuan Hana, Yun Jianga, Guijuan Yangd , Jing Tiana, Qin Maa, Yi Zhanga, *

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a

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University, Yangling, Shaanxi, 712100, P.R China

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b

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Paulownia R&D Center of State Administration of Forestry and Grassland, Zhengzhou, Henan 450003, China

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c

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d

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State Key Laboratory of Crop Stress Biology in Arid Areas, College of Forestry, Northwest A&F

Department of Computer Science and Bioinformatics, Khushal Khan Khattak University, Karak, Khyber Pakhtunkhwa 27200, Pakistan

Key Laboratory of Tree Breeding and Cultivation of State Forestry Administration, Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China

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*

Corresponding author

E-mail address: [email protected]

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Can Yang and Wenwen Qiu are equal contributors

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Keywords: Stem development, Cadmium, Antioxidant defense, Phosphorus status, N:P stoichiometric

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ratio, Populus,

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Abstract

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The soils in mining lands with cadmium (Cd) contamination usually are deficient in nutrients.

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Disclosing how P nutrition and N:P stoichiometric ratio influences Cd accumulation and stress tolerance

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in stems of Populus spp. will facilitate the phytoremediation of mining sites polluted by Cd. In this study,

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investigations at the anatomical and physiological levels were conducted using a clone of Populus ×

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euramericana. Both phosphorus deficiency and cadmium exposure inhibited xylem development via

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reducing cell layers in the xylem. Under P-sufficient condition, appropriate P status and balanced N:P

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ratio in stem promoted xylem development under Cd exposure via stimulating cell division, which

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enhanced Cd accumulation in stems. Cd accumulation in cell walls of collenchyma tissues of the stem

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was enhanced by P application due to increased polysaccharide production and cell wall affinity for Cd.

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The low P concentrations (0.3~0.4 mg g-1) and imbalanced N:P ratio under P deficiency inhibited the

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production of APX and ascorbate–GSH cycle, which increased oxidative stress and lipid peroxidation as

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indicated by high MDA concentration in stem. Under P-sufficient condition, the interactions between

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phytohormones and antioxidants play crucial roles in the process of antioxidant defense under Cd

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exposure. In conclusions, appropriate P addition and balanced N:P ratio enhanced secondary xylem

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development and promoted cadmium accumulation and stress tolerance in Populus stems, which can

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benefit the phytoextraction of Cd from Cd-contaminated soil.

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Keywords: Stem development, Cadmium, Antioxidant defense, Phosphorus status, N:P stoichiometric

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ratio, Populus

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Introduction

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Environmental contamination by toxic heavy metals is increasing due to increasing human activities

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such as mining (He et al., 2013, 2015; Shi et al., 2015). Long-term mining is a major resource of heavy

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metal contamination and health risk to humans (Liu et al., 2014). Mining activities in China alone have

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generated about 3.0 million ha derelict land by the year 2000, and it is increasing at a rate of 46,700 ha

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per year (Li 2006). The food security has been challenged dramatically with the loss of the arable lands

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in China (He et al., 2011; Liu et al., 2014). Therefore, ecological restoration of mining sites and

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phytoremediation of heavy metal contamination is critical to reduce health risk and enhance food

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security in China. After metal mining, the mining lands usually have inferior soil properties that are

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unfavorable to plant establishment, such as the deficiency of nutrients, poor physical structure and

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extreme pH (Li 2006; Liu et al., 2014; Rees et al., 2016). To break through the constraints of nutrient

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deficiency to ecological restoration of mining sites with cadmium contamination, it is necessary to

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elucidate the mechanisms underlying the impact of nutrient status on cadmium accumulation and stress

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tolerance in plant species that are used for ecological restoration (Hacke et al., 2010; Zhang et al., 2019).

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Cadmium (Cd) is a widespread heavy metal in mining lands that lead to pollution of soils and waters,

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and endanger the health of humans (Liu et al., 2013; Shi et al., 2015). Cd in the soil can be extracted by

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metal-tolerant plant species through absorption by roots, sequestering into the vacuole as Cd-

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phytochelatin complex, and translocation into shoots (Farooq et al., 2013; Chen et al., 2016; Cheng et al.,

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2017). Phosphorus (P) is an essential macronutrient which not only support plant growth but also

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reduce Cd toxicity via chelating or forming complex with Cd in plants, which may alleviate the damage

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of Cd to cell functions (Jiang et al., 2007; Gomes et al., 2014; Dai et al., 2017a). P addition decreased

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malonaldehyde via positively regulating the synthesis of glutathione and phytochelatins in mangrove

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plants (Dai et al., 2017a). The growth of Mirabilis jalapa L increased with P supplies under Cd exposure, 2

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and the translocation factor of Cd reached the maximum at 100 mg kg−1 P application (Dai et al., 2017a).

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However, the anatomical and physiological mechanisms underlying the influence of phosphorus on Cd

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accumulation and antioxidant defense in plant species with high Cd tolerance and large capacity for Cd

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phytoextraction are still not fully elucidated.

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Cadmium hyperaccumulator plants have been proposed for the phytoremediation of Cd-contaminated

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mining sites as they are highly tolerant to this toxic metal and can absorb and accumulate large

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concentration of Cd from contaminated soils (McGrath et al., 2006; Liu et al., 2017; Wu et al., 2018).

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Some fast-growing woody species such as Populus spp. also showed high tolerance to Cd exposure

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(Chen et al., 2011; He et al., 2013, 2015; Shi et al., 2015). Although the tissue concentration of Cd in

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these woody species may be lower than herbaceous hyperaccumulators, some Populus species usually

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extract a similar or even higher total amount of Cd from contaminated soil due to their gigantic root

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system and great biomass (Wu et al., 2010; Chen et al., 2011; Zhang et al., 2019; Wang et al., 2019). To

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alleviate oxidative stress and avoid damages caused by Cd exposure, Populus species usually exhibit

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various adaptations such as the inducement of enzymatic and non-enzymatic antioxidants that are critical

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to the homeostasis of ROS production and scavenging (Seth et al., 2012; Shi et al., 2015; Redovniković

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et al., 2017; Zhang et al., 2019; Wang et al., 2019).

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Once Cd ions are in the xylem of the roots, they pass up the xylem of the stem and transported to the

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leaves with the transpiration stream, or be re-distributed via phloem transport (He et al., 2015; Wang et

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al., 2019). The xylem-mediated Cd translocation into shoots is a major factor determining Cd

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accumulation in the shoots of many plants, and the disruption or wound of xylem may results in reduced

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Cd accumulation in above-ground tissues (Qin et al., 2013; Wang et al., 2019). There was an 80%

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reduction in stem diameter Eucalyptus grandis with decreasing P supply, all of which can be directly

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attributable to the reduction in cell numbers, as new cells require P for nucleic acids, membranes and 3

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energy supply during construction (Thomas et al., 2005). Previous research demonstrated that poplar

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species might respond to P deficiency by decreasing P uptake, mobilization and assimilation (Gan et al.,

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2015). Together with nitrogen, phosphorus is a key component of the metabolites involved in

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photosynthesis and the production of carbohydrates as well as antioxidants that are critical to sustaining

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antioxidative processes (Jiang et al., 2007; Rennenberg et al., 2013; Cheng et al., 2018). Moreover, plant

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growth is determined by interactions among nutrients, particularly N:P ratios in ecosystems and plant

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tissues, i.e. the relative availability of environmental N and P (Hu et al. 2016; Shi et al., 2017).

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Therefore, disclosing how P nutritional status and N:P stoichiometric ratio influences stem development

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and antioxidant defense, and finally affecting Cd accumulation in stems of Populus spp. will facilitate

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the phytoremediation of mining sites polluted by heavy metals.

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In this study, investigations at the anatomical and physiological levels were conducted using a clone

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of Populus × euramericana to address the following hypotheses: (1) P status and N:P stoichiometric

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ratio will influence the secondary xylem development and the production of enzymatic and non-

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enzymatic antioxidants in stems of poplar under combined conditions of P and Cd. (2) P nutritional

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status and N:P stoichiometric ratio will influence Cd accumulation in stems via regulating the secondary

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xylem development and antioxidant defense. This study will provide important plant nutritional

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guidelines for the phytoremediation of mining sites polluted by heavy metals using poplar species.

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Materials and methods

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Plant material

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A hybrid poplar species, Populus × euramericana which showed favorable tolerance to heavy metal,

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drought and salt stress (Wu et al., 2010; Di Baccio et al., 2011) was used as the plant material. Plantlets

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were produced by micropropagation and cultivated in a climate chamber (day/night temperature, 4

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25/18 °C; relative air humidity, 50–60%; light per day, 14 h; photosynthetic photon flux, 100 µmol m–2

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s–1). After growing for 5 weeks, rooted plantlets were transferred to 10-L plastic pots filled with fine

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sand. Subsequently, plants were grown for 4 weeks in a greenhouse (day/night temperature, 25/18 °C;

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relative air humidity, 50–60%; light per day, natural light). After growing for 4 weeks, plants with

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similar height were selected and divided into several groups for further treatments.

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Experimental design

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A factorial design consisting of P and Cd treatments was applied. There were three Cd levels and two P

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levels in the factorial design. The three Cd levels were 0 mg Cd kg-1 sand, 4 mg Cd kg-1 sand and 20 mg

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Cd kg-1 sand respectively, which were achieved by adding 0, 7.4, or 37.0 µg g−1 CdSO4 into the sand in

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the form of a solution. Modified LA solution (Luo et al., 2013) with normal P and deficient P nutrient

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was applied to pots of the control and the deficient-P treatment, respectively. Phosphorus concentrations

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in deficient P treatment (0.06 mM KH2PO4, 0.0042 mM K2HPO4) was equal to 10% of phosphorus

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concentrations in the control (0.6 mM KH2PO4, 0.042 mM K2HPO4), while the nutrient concentrations

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except P (1.0mM NH4NO3, 0.5 mM KCl, 0.9 mM CaCl2, 0.3 mM MgSO4, 10 µM Fe-EDTA, 2 µM

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MnSO4, 10 µM H3BO3, 7 µM Na2MoO4, 0.05 µM CoSO4, 0.2 µM ZnSO4, and 0.2 µM CuSO4) were

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same for the control and low P treatments. Although deficient P treatment had a low concentration (0.06

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mM) of KH2PO4, the plants in deficient P treatment can obtain adequate K+ as 0.5 mM KCl was equally

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applied in both P treatments. Therefore, the influence of K+ deficiency on Cd accumulation and Cd

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tolerance can be neglected. The nutrient solution was applied at a rate of 200 mL per pot every day.

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There were 9 replicates in each treatment, giving a total of 54 seedlings. The P and Cd treatments were

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applied for 60 days until harvest. The total time of plantlets growth was 88 days.

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Measurements of seedling growth traits and tissue P and N concentration

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At harvest, plant height, stem basal diameter and fresh weight of each organ were measured and then

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frozen immediately in liquid nitrogen. The frozen samples were ground into a fine powder, during which

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the liquid nitrogen was applied to maintain the freezing status. The fine powder of each sample was

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stored at −80 °C until further analysis. The fresh : dry mass ratio were determined via drying the fresh

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sample (about 100 mg) at 65 °C for 72 h and measuring the dry biomass (Gan et al., 2015). The biomass

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of each organ was determined using the fresh weight and the fresh : dry mass ratio. Root to shoot ratios

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(R/S) was calculated as the ratio of root biomass to shoot biomass. To determine the concentrations of P

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and N in each organ, approx. 100 mg sample were digested, and subsequently, the P concentration and

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N concentration in the digested solution was determined according to the method described by previous

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reports (Gan et al., 2015).

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Determination of tissue distribution of Cd and anatomical features of stem

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To determine the patterns of localization and distribution Cd in stems, histochemical staining of Cd was

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conducted using fresh samples from poplar seedlings grown under high Cd treatment (20 mg Cd kg-1 soil)

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and control (0 mg Cd kg-1 soil). Sections of the stems (ca. 10 cm long below the top of stem) were

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rinsed in de-ionized H2O. Subsequently, fresh samples of stems were immersed in a staining solution for

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1 h and then rinsed briefly in de-ionized H2O (He et al., 2015). Subsequently, the well-stained sections

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with Cd-dithizone precipitates were photographed under a light microscope (Eclipse E200; Nikon,

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Tokyo, Japan) using a CCD (DS-Fi1; Nikon) connected to a computer, as described by previous reports

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(He et al., 2015). The stained sections of stems were photographed to perform anatomical analysis. Fiber

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lumen diameters, xylem cell layers, and thicknesses of xylem and phloem were quantified using the

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method described by previous studies (Song et al., 2019).

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Determination of Cd accumulation and Cd bioconcentration factor in stems

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To measure Cd content in stems, fine powder (ca. 100 mg) of samples was digested at 170◦C in a

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mixture of acid (7 ml HNO3 and 1 ml concentrated HClO4). Subsequently, Cd was determined by flame

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atomic absorbance spectrometry (Hitachi 180-80, Japan). Cadmium concentration was measured using

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flame atomic absorbance spectrometry (Hitachi 180-80; Hitachi Ltd, Tokyo, Japan) according to the

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reported method (He et al., 2015). Cadmium content was calculated by multiplying the Cd concentration

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in each organ by the organ biomass. Bioconcentration factor (BCF) was calculated as the ratio of Cd

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concentration in an organ to the Cd concentration in the soil (He et al., 2013).

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Measurement of the N and P concentration and N:P stoichiometric ratio

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For the measurement of physiological parameters related to stress tolerance, fresh samples from three

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seedlings were incorporated to generate a mixed sample, resulting in three biological replicates in each

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treatment which consisting of 9 seedlings. The dried plant organs were used for measurement of the N

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and P concentration, using a method described by Gan et al. (2015). Fine powder (∼100 mg DW) was

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digested by 5 ml 98% H2SO4 and 1 ml H2O2. The P concentration was determined

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spectrophotometrically at 700 nm based on the molybdenum blue method. The N concentrations were

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analyzed using a Continuous-Flow Analyzer (AA3, Bran-Luebbe, Hamburg, Germany) at 660 nm. The

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N:P stoichiometric ratio was calculated as tissue N concentration divided by tissue P concentration.

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Physiological parameters related to stress tolerance in stems

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The concentration of soluble sugars was determined using the previous method (Luo et al., 2013). The

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concentration of free proline was determined using the following method. Fresh samples (ca. 100 mg)

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was extracted by 1.5 ml of 3% sulfosalicylic acid at 100 °C for 15 min, and then they were centrifugated

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for 10 min at 12000 g. Subsequently, 1 mL glacial acetic acid and 1 mL ninhydrin reagent was mixed

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with the supernatant and then incubated at 98 °C for 30 min. Finally, the solution were

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spectrophotometrically measured at 518 nm. Using methods reported by Chen et al. (2011), the activities 7

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of antioxidant enzymes were determined, which including peroxidase (POD; EC 1.11.1.7), ascorbate

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peroxidase (APX; EC 1.11.1.11), catalase (CAT; E.C. 1.11.1.6) and glutathione reductase (GR; EC

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1.6.4.2). Malonaldehyde (MDA) concentration was determined based on the method previously reported

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(He et al., 2013).

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Phytohormone levels in stems including jasmonic acid (JA), abscisic acid (ABA), gibberellic acid

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(GA3), and salicylic acid (SA) were analyzed through high-performance liquid chromatography-

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electrospray ionization-ion trap mass spectrometry. Briefly, 4 mL of 80% (v/v) methanol containing 200

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mg µL−1 of butylated hydroxytoluene and 500 mg L−1 of citric acid monohydrate was used to extract

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phytohormones from the powder of fresh samples (ca. 500 mg). The extracting solution was shaken at

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4 °C for 12 h, and then centrifugated at 10,000 g and 4 °C for 15 min. The supernatant was collected,

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and the precipitate was extracted twice using the same method. The supernatants were incorporated and

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dried by N2. Before measurement, the dried samples were re-suspended by 800 µL of 80% methanol,

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and were analyzed by an LC-20AT high-performance liquid chromatography system (Shimadzu, Kinh

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Do, Japan) combined with an API 2000™ electrospray tandem mass spectrometer (Allen-Bradley,

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Milwaukee, WI, USA). ABA [(±)-ABA, A1049], GA3 (G7645), SA (S7401), and IAA (I2886) were

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used to make standard curves.

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Statistical analysis

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The normality of data was tested before statistical analyses using the UNIVARIATE procedure of SAS

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software (SAS Institute, Cary, NC; 1996). Statistical tests were performed with SAS software (SAS

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Institute, Cary, NC; 1996). The P-values of multiple comparisons were calculated using Tukey’s HSD

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method to avoid type I errors (He et al., 2015). Differences between values were considered to be

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significant when the P-value of the ANOVA F-test was < 0.05. Linear correlations were analyzed by the

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CORR procedure in the SAS software (SAS Institute, Cary, NC; 1996), and were considered to be 8

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significant when the P-value was less than 0.05. The principal component analysis (PCA) were

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performed by the command prcomp() in R (http://www.rproject.org/) as reported previously (Zheng et

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al., 2017).

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Results

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Biomass and anatomical features of stem

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Plant biomass, plant height, stem basal diameter and stem biomass were decreased by P deficiency

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regardless of Cd treatments, while root biomass was unaltered by P status(Fig. 1). As a result, R/S was

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increased by P deficiency. Plant biomass and stem biomass were decreased by Cd exposure under both

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deficient- and normal- P conditions, while root biomass was unaltered by Cd exposure. R/S increased

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upon Cd exposure under both P conditions (Fig. 1). The degree of suppression on plant biomass and

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stem biomass was the largest under the combination of Cd exposure and P deficiency (Fig. 1)

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Phosphorus and cadmium had significant effects on xylem thickness and xylem cell layers. Cd

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exposure suppressed xylem development under both P deficiency and control, resulting in the lower

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thickness and less cell layers of xylem (Fig. 2 a-d; Fig. 2 i-j). Xylem development was suppressed by P

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deficiency under high or zero Cd exposure. The degree of suppression on xylem development was the

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largest under the combination of Cd exposure and P deficiency, while the suppression was partially

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alleviated by P application (Fig. 2) (Fig. 2 a-d; Fig. 2 i-j). The suppression on xylem development was a

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result of decreased cell layers of xylem but not fiber cell size, as fiber size was unaltered by P deficiency

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or Cd exposure (Fig. 2 i-j).

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Accumulation and bioconcentration of Cd and its correlation with xylem development

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In stems, Cd was located mainly in the symplast of collenchyma cells (Fig. 2h) when soil P was

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deficient. In contrast, Cd was distributed more widely and intensely under normal P condition than that

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of low P status, and it was located in symplast and cell walls of collenchyma cells, as well as in apoplast 9

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and intercellular space of cortical cells (Fig. 2f). The concentration of Cd in stems were decreased by P

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deficiency, which decrased from approx. 12 µg mg-1 to 4 µg mg-1, and from approx. 18 µg mg-1 to 12 µg

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mg-1 under moderate and high Cd exposure, respectively (Fig. 3). As a result, Cd content in stem

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decreased from approx. 120 µg plant-1 to 20 µg plant-1, and from approx. 160 µg plant-1 to 60 µg plant-1

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under moderate and high Cd exposure, respectively (Fig. 3). The bioconcentration factor (BCF) of Cd in

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stems was decreased by P deficiency from approx. 3.0 to 1.0 under moderate Cd expsure, while the

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changes by P application was not significant under high Cd exposure (Fig. 3). Under high Cd exposure,

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the concentration of Cd in stems was positively correlated with the xylem thickness and the number of

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cell layers, with the coefficient of determination (R2) being 0.84 and 0.75, respectively (Fig. S1).

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Phosphorus status, N:P ratios and their correlations with Cd accumulation and xylem development

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In stems, P concentrations sharply decreased upon soil P deficiency regardless of Cd level, decreased

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from around 1.8~2.0 mg g-1 under normal P condition to around 0.3~0.4 mg g-1 under P deficiency. In

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comparison, tissue N concentrations were not altered by soil P conditions at each Cd level (Table 1). As

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a result, tissue N : P ratios in stems sharply increased from around 4.5~6.5 under normal P condition to

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around 17.0~23.0 under P deficiency (Table 1). Under both P conditions, concentrations of P, N and N :

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P ratios in stems all were unaltered by Cd exposure (Table 1).

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Under high Cd exposure, P concentration in both roots and stems had a positive correlation with

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xylem thickness and cell layers in xylem (Table S1). In contrast, N:P ratios in both roots and stems had a

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negative correlation with the thickness and cell layers of xylem. Phosphorus concentration in both roots

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and stems had a strong and positive correlation with the concentration and content of Cd in stem. The

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trend of N : P ratio was opposite to that of P concentration, as N : P ratio in both stems and roots are

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negatively correlated with Cd concentration and Cd content in stems (Table S1).

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Concentrations of free proline, soluble sugars and soluble protein in stems 10

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Under normal P conditions, the level of free proline in stems was increased by a high or moderate level

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of Cd exposure as compared with zero Cd exposure (Table S2). In low-P treatment, the levels of free

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proline, soluble sugars and soluble proteins in stems were all unaltered by Cd exposure. Under the high

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or moderate level of Cd exposure, the level of free proline in stems decreased upon P deficiency (Table

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S2). The levels of soluble sugars and soluble protein were unaltered by P deficiency at each Cd level,.

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The levels of antioxidant enzymes, GSH and MDA in stems

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Under normal P condition, GSH concentration and the activities of APX and POD all increased upon

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high Cd exposure, while all antioxidant enzymes were unaltered by moderate Cd exposure (Fig. 4; Fig.

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S2). Under P deficient condition, POD was stimulated by both moderate- and high- Cd exposure (Fig. 4),

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while GR and CAT activity increased upon moderate- and high- Cd exposure, respectively. GR and

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CAT activity increased upon P deficiency at a moderate and high level of Cd exposure, respectively.

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The activities of the other enzymes were unaltered by P status regardless of Cd level (Fig. 4).

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MDA level in the stems was higher under low P conditions than that of control at each Cd level. High

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Cd exposure increased MDA level under deficient P condition, but not under normal P condition (Fig.

242

S3). MDA concentration in stem is negatively correlated with xylem thickness (R2=0.79) and the cell

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layers of xylem (R2=0.81) (Fig. S4).

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The levels of phytohormones in stems

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Under normal P condition, ABA was unaltered by Cd exposure, while JA was decreased by moderate or

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high level of Cd exposure. In comparison, GA3 was increased by both moderate- and high- Cd exposure,

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and SA was increased by high Cd exposure (Fig. 5). Under P-deficient condition, all of the tested

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phytohormones (ABA, JA, GA3 and SA) were decreased by a high level of Cd exposure, and they were

249

also decreased by moderate Cd exposure except SA (Fig. 5).

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The levels of ABA, GA3 and SA were increased by P deficiency in the absence of Cd exposure. In

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contrast, the levels of JA and SA decreased upon P deficiency under moderate- and high Cd exposure,

252

respectively. The levels of GA3 decreased upon P deficiency at both Cd exposure levels (Fig. 5).

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PCA of physiological responses to Cd stress under different N conditions

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To display the general trends of growth and physiological traits, and the patterns of cadmium

255

accumulation and antioxidant defense under contrasting P conditions, PCA was performed based on

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growth and root traits, anatomical features of stem, Cd accumulations in roots and stems, phosphorus

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concentrations and N:P ratios, and the levels of antioxidants and phytohormones. PC1 and PC2

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accounted for 33.7% and 14.5% of the variation, respectively (Fig. 6). In general, the effects of P

259

conditions were separated by PC1, while the effects of Cd exposure were jointly separated by PC1 and

260

PC2. Dominant variables for PC1 were stem biomass, root N:P ratio, stem P concentration, stem N:P

261

ratio, stem MDA concentration, stem GR activity, root ABA concentration, aboveground biomass, plant

262

R/S, plant biomass (Table S3). The key factors of PC2 were activities of antioxidant enzymes APX and

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POD in stem, the levels of phytohormones IAA ABA and JA in stems, root biomass and thick root

264

length, Cd concentration and content in stems (Table S3).

265

Discussion

266

Growth traits and stem anatomical features were affected by combined conditions of P and Cd

267

Both phosphorus deficiency and Cd exposure suppressed plant height and biomass production, while the

268

degree of suppression on xylem development was the largest under the combination of Cd exposure and

269

P deficiency. Stem P concentration was not inhibited by Cd exposure, indicating that the absorption of P

270

was not sensitive to Cd stress. Research in Atriplex atacamensis showed that arsenate decreased the P

271

concentrations in a less extent than for P starvation (Vromman et al., 2017). Therefore, it can be

12

272

demonstrated that P absorption by plants are more sensitive to soil P deficiency and less sensitive to

273

heavy metal exposure such as Cd and As.

274

Phosphorus is essential for nucleic acids, membranes and energy supply during cell division and

275

construction (Thomas et al., 2006; Gan et al., 2016). The present study indicated that both phosphorus

276

deficiency and cadmium exposure inhibit xylem development via reducing cell layers in the xylem. The

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degree of suppression on xylem development was the largest under the combination of Cd exposure and

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P deficiency, and the growth suppression was partially alleviated under the combination of Cd exposure

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and normal P condition. Stem P concentrations (0.3~0.4 mg g-1) was extremely low under P deficiency,

280

and it was approx. four times lower than the mean tissue P concentration (1.45 mg g-1) of China’s flora

281

(Wu et al., 2012). In comparison, it was appropriate (1.8~2.0 mg g-1) under normal P condition.

282

Correlation analysis revealed that stem P concentration was positively correlated with xylem thickness

283

and xylem cell layers. It can be concluded that appropriate P status in stem promoted xylem

284

development under high Cd exposure, while the low tissue P concentration increased the degree of

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growth suppression under Cd exposure due to the reduced cell numbers.

286

Nitrogen and phosphorus stoichiometry plays vital roles in studying nutrient limitation (Wu et al.,

287

2012). It was proposed that a leaf N:P < 14 indicated N limitation, and leaf N:P > 16 indicated P

288

limitation. The optimal value of N:P ratio in roots or stems is usually lower than that in leaf, because

289

nitrogen demand by the leaf is higher than that by stem due to the photosynthesis function in leaf (Gan

290

et al., 2015; Yan et al., 2016). The concentration of N and P in roots Populus simonii Carr under normal

291

nutrient conditions was approx. 15.0 mg g-1 and 3.0 mg g-1 respectively (Gan et al. 2015), implying that

292

a N:P ratio of 5.0 can be suitable for poplar roots or stems. In this study, stem N:P ratios sharply

293

increased from 4.5~4.9 under normal P condition to around 21~23 under P deficiency, indicating a

294

severe P-limitation under deficient-P condition (Wu et al. 2012). Further analysis indicated that N:P 13

295

ratios in stems had a negative correlation with xylem thickness and xylem cell layers. These results

296

demonstrated that the poor P nutritional status and imbalanced N:P ratio in stems should be responsible

297

for the severe suppression of xylem development under the combined condition of Cd exposure and P

298

deficiency. In contrast, appropriate P status and balanced N:P ratio alleviated growth suppression under

299

high Cd exposure, due to active cell division and construction in stem with adequate supply of nucleic

300

acids, membranes components and energy under normal P condition (Luo et al., 2009; Gan et al., 2015).

301

Phosphorus application promoted Cd accumulation in stems via stimulating xylem development

302

Cd is transported from roots to shoots mainly via the xylem, and thus the xylem structure may influence

303

Cd transport and Cd accumulation in shoots (Qin et al., 2013). Xylem removal dramatically inhibit Cd

304

transportation from roots to above-ground tissues and thus reduce Cd accumulation in shoots (Qin et al.,

305

2013). In the present study, xylem development was markedly promoted by adequate P application as

306

indicated by increased xylem thickness and xylem cell layers. Taken together with the positive

307

correlation between Cd concentration in stems and xylem development, we can deduce that normal P

308

application stimulated cell division and construction of xylem and thus promoted Cd translocation and

309

Cd accumulation in stems. In contrast, the poor P nutritional status and imbalanced N:P ratio under P

310

deficient condition lead to a severe suppression of xylem development under Cd exposure, which

311

inhibited Cd accumulation and Cd bioconcentration in stems.

312

In addition to affecting Cd translocation, phosphorus can also influence rates of Cd fixation in the cell

313

wall. For example, P deficiency significantly decreased the amount of Cd retained in the root cell wall of

314

Arabidopsis thaliana (Zhu et al., 2012), whereas P application increased Cd concentrations in roots of

315

mangrove seedling (Dai et al., 2017b). In the present study, Cd concentration in poplar stems was higher

316

under normal P conditions as compared with P deficiency (Fig. 2). In particularly, Cd distribution in cell

317

walls of collenchyma tissues was far more outstanding under normal P conditions than that in P 14

318

deficiency, suggesting that exogenous P increased cell wall affinity for Cd and lead to greater Cd

319

accumulation in the cell wall. It was demonstrated that phosphorus might increase Cd accumulation in

320

cell walls via increasing cell wall pectin and hemicellulose 1 concentrations (Dai et al., 2017b). In the

321

present study, the outstanding accumulation of Cd in cell walls of stem collenchyma tissues may be

322

explained as the enhanced capacity of cell walls for Cd binding due to the increasing polysaccharide

323

production with sufficient P application. Moreover, P application may promote the pectin content and

324

PME activity, which can generate free carboxyl groups available for Cd binding in the cell wall (Dai et

325

al., 2017a). Further analysis revealed that N:P ratios in both stems and roots are negatively correlated

326

with Cd concentration and Cd content in stems, indicating an increasing biological capacity for Cd

327

accumulation and stress tolerance in poplar stems with sufficient P supply and balanced N:P

328

stoichiometry ratio. Additionally, Cd accumulation increased with the increasing intensity of Cd

329

exposure. These results demonstrated that the poplar clone used in this study has strong potential for

330

phytoremediation Cd pollution with sufficient P supply.

331

P application and balanced N:P ratio promoted Cd tolerance via enhancing antioxidant defense

332

Cadmium exposure can lead to concentration-dependent oxidative stress in plants, while plants can

333

combat oxidative stress via inducing the antioxidant systems (He et al., 2015; Shi et al., 2015). Several

334

antioxidant enzymes participate in the scavenging of ROS (reactive oxygen species) such as O2•− and

335

H2O2 (Ma et al., 2013; He et al., 2015). POD play a crucial role in the scavenging of H2O2 and hydroxyl

336

radicals (Zhang et al., 2019). In the present study, the activity of POD was increased by high Cd

337

exposure regardless of soil P conditions. Therefore, the role of POD in Cd tolerance was independent of

338

P conditions in the present study. APX and GR participated in the form of ascorbate–GSH cycle that is

339

responsible for scavenging H2O2 under stress (He et al., 2015; Ma et al., 2018; Zhang et al., 2019). In the

340

present study, the activity of APX was increased by high Cd exposure when soil P was sufficient, while 15

341

this induction was not observed under P deficiency. A previous study in Atriplex atacamensis indicated

342

that glutathione reductase (EC 1.6.4.2) is affected in some way by P depletion, which will lead to higher

343

sensitivity to As (Vromman et al, 2017). Our results indicated that the extremely low P concentrations

344

(0.3~0.4 mg g-1) and unbalanced N:P ratio under P deficiency can lead to the absent induction of APX

345

and ascorbate–GSH cycle, which should contributed to the increased oxidative stress and lipid

346

peroxidation as indicated by high MDA concentration. Moreover, MDA concentration in a stem is

347

negatively correlated with xylem thickness and cell layers, indicating that increasing MDA level and

348

oxidative stress can lead to greater inhibition on xylem development.

349

In addition to antioxidant enzymes, phytohormones such as ABA, SA and JA also play crucial roles

350

in combating oxidative stress under heavy metal exposure (H et al., 2011; He et al., 2015; Zhang et al.,

351

2019). Previous studies reported diverse patterns of phytohormone inducement under heavy metal

352

exposure, which may vary across plant species and/or environmental factors (Sofo et al., 2013; Shi et al.,

353

2015; Wang et al., 2019). Under normal P condition in the present study, GA3 and SA were increased by

354

high Cd exposure. However, all of the tested phytohormones (ABA, JA, GA3 and SA) were decreased

355

by Cd exposure under P deficiency. It was reported that although of a variety of antioxidants can combat

356

Cd stress, severe Cd stress may also conversely disturb or interfere with the biosynthesis of these

357

antioxidants including phytohormone, GSH and PC (Zhu et al., 2012; Wang et al., 2019). Our results

358

indicated that the inducement of phytohormones upon Cd exposure were highly related to P status.

359

When soil P was deficient, the biosynthesis of various phytohormones were inhibited by high Cd

360

exposure, presumably due to the increasing oxidative stress as a result of the absent induction of

361

antioxidant enzymes such as APX and GR under P deficiency. The patterns revealed by the present

362

study can be considered as a negative “cross-talk” between phytohormones and antioxidant enzymes

363

when soil P deficiency and Cd exposure synchronously occurs. 16

364

Under normal P condition, the concentrations of some specific phytohormones (SA and GA3) were

365

promoted by high Cd exposure. It was reported that SA can stimulate N and S metabolism, and thus

366

increase the levels of APX and GR, which can contribute to stress tolerance and growth inhibition under

367

stress (Nazar et al., 2011; Wang et al., 2019). Interestingly, a sharp inducement of antioxidase activity

368

(APX, GR and POD) upon Cd exposure was also observed under normal P conditions of the present

369

study. These findings implying the interactions between phytohormones and antioxidants can play

370

crucial roles in the process of antioxidant defense under Cd exposure (He et al., 2015), which at least

371

partially dependent on normal P nutritional conditions and balanced N:P ratio.

372

Conclusions

373

The mechanisms underlying the influence of phosphorus status and N:P stoichiometric ratio on cadmium

374

accumulation and stress tolerance in Populus stems were explored (Fig. 7). Under P-sufficient condition,

375

sufficient tissue P concentration and balanced N:P ratio enhanced cell division and xylem construction

376

and thus promoted Cd translocation and increased Cd accumulation in stems. In contrast, the extremely

377

low tissue P concentration and imbalanced N:P ratio under P deficiency down-regulated cell divisions

378

and reduced cell numbers and cell layers, which may inhibit Cd Cd accumulation in stems. Phosphorus

379

addition increased cell wall affinity for Cd and enhanced Cd accumulation in stem cell wall, presumably

380

due to active polysaccharide production after P application. The low P concentrations and imbalanced

381

N:P ratio under P deficiency inhibited APX production and ascorbate–GSH cycle, which lead to

382

increasing oxidative stress in stem. A negative interaction between phytohormones and antioxidant

383

enzymes was detected in poplar stems when soil P deficiency and Cd exposure synchronously occur. In

384

contrast, the positive interactions between phytohormones and antioxidants was crucial for antioxidant

385

defense under normal P conditions. In conclusions, appropriate P addition and balanced N:P ratio can

17

386

enhance phytoextraction of Cd from Cd-contaminated soil via enhancing secondary xylem development,

387

up-regulating antioxidant defense and promoting cell wall affinity for Cd in Populus stems.

388 389

Acknowledgements

390

This project was financially supported by National Key R&D Program of China (Grant No.

391

2016YFD0600105), the Start-up Project for Introduced Recruit in Northwest A&F University

392

(Z111021402), the Fundamental Research Funds for the Central Universities (No. 2452016056), and the

393

China Scholarship Council (CSC NO. 201706305011).

394 395

Appendix A. Supporting information

396

Supplementary data associated with this article can be found in the online version

397

References

398

Chen, L., Han, Y., Jiang, H., Korpelainen, H., Li, C., 2011. Nitrogen nutrient status induces sexual differences in

399 400 401 402 403

responses to cadmium in Populus yunnanensis. J. Exp. Bot. 62 (14),5037–5050. Chen, L.H., Zhang, D., Yang, W.Q., Liu, Y., Gao, S., 2016. Sex-specific responses of Populus deltoides to Glomus intraradices colonization and Cd pollution. Chemosphere 155,196–206. Cheng, M.M., Wang, A., Tang, C.X., 2017. Ammonium-based fertilizers enhance Cd accumulation in Carpobrotus rossii grown in two soils differing in pH. Chemosphere 188,689–696.

404

Cheng, Y., Wang, C., Chai, S., Shuai, W., Wang, Y., 2018. Ammonium N influences the uptakes, translocations,

405

subcellular distributions and chemical forms of Cd and Zn to mediate the Cd/Zn interactions in dwarf polish wheat

406

(Triticum polonicum L.) seedlings. Chemosphere 193, 1164–1171.

407

Dai, M.Y., Lu, H.L., Liu, W.W., Jia, H., Hong, H.L., Liu, J.C., Yan, C.L., 2017a. Phosphorus mediation of cadmium

408

stress in two mangrove seedlings Avicennia marina and Kandelia obovata differing in cadmium accumulation.

409

Ecotox. Environ. Safe. 139, 272–279.

18

410

Dai, M.Y., Liu, J.C., Liu, W.W., Lu, H.L., Jia, H., Hong, H.L., Yan, C.L., 2017b. Phosphorus effects on radial oxygen

411

loss, root porosity and iron plaque in two mangrove seedlings under cadmium stress. Mar.Pollut. Bull. 119, 262–

412

269.

413 414

Di Baccio, D., Galla, G., Bracci, T., Andreucci, A., Barcaccia, G., Tognetti, R., Sebastiani, L., 2011. Transcriptome analyses of Populus x euramericana clone I-214 leaves exposed to excess zinc. Tree Physiol. 31(12), 1293–1308.

415

Farooq, M.A., Ali, S., Hameed, A., Ishaque, W., Mahmood, K., Iqbal, Z., 2013. Alleviation of cadmium toxicity by

416

silicon is related to elevated photosynthesis, antioxidant enzymes; suppressed cadmium uptake and oxidative stress

417

in cotton. Ecotox. Environ. Safe. 96,242–249.

418

Gan, H.H., Jiao, Y., Jia, J.B., Wang, X., Li, H., Shi, W.G., Peng, C.H., Polle, A., Luo, Z.B., 2016. Phosphorus and

419

nitrogen physiology of two contrasting poplar genotypes when exposed to phosphorus and/or nitrogen starvation.

420

Tree Physiol. 36, 22–38.

421 422

Gomes, M.P., Soares, A.M., Garcia, Q.S., 2014. Phosphorous and sulfur nutrition modulate antioxidant defenses in Myracrodruom urundeuva plants exposed to arsenic. J. Hazard. Mater. 276, 97–104.

423

Hacke, U.G., Plavcová, L., Almeida-Rodriguez, A., King-Jones, S., Zhou, W.C., Cooke, J.E.K., 2010. Influence of

424

nitrogen fertilization on xylem traits and aquaporin expression in stems of hybrid poplar. Tree Physiol. 30,1016–25.

425

He, J., Ma, C., Ma, Y., Li, H., , Kang, J., Liu, T., Polle, A., Peng, C., Luo, Z.B., 2013. Cadmium tolerance in six

426

poplar species. Environ. Sci. Pollut. R. 20, 163–174.

427

He, J., Li, H., Ma, C.F., Zhang, Y., Polle, A., Rennenberg, H., Cheng, X., Luo, Z., 2015. Overexpression of bacterial c-

428

glutamylcysteine synthetase mediates changes in cadmium influx, allocation and detoxification in poplar. New

429

Phytol. 205, 240–254.

430

He, J., Qin, J., Long, L., Ma, Y., Li, H., Li, K., Jiang, X., Liu, T., Polle, A., Liang, Z., Luo, Z.B., 2011. Net cadmium

431

flux and accumulation reveal tissue-specific oxidative stress and detoxification in Populus×canescens. Physiol.

432

Plantarum. 143, 50–63.

433 434

Hu, C.Y., Zhao, L., Zhou, Z.C., Dong, G.Q., Zhang, Y., 2016. Genetic variations and correlation analysis of N and P traits in Pinus massoniana under combined conditions of N deposition and P deficiency. Trees 30, 1341–1350.

19

435 436 437 438 439 440 441 442 443 444 445 446

Jain, M., Pal, M., Gupta, P., Gadre, R., 2007. Effect of cadmium on chlorophyll biosynthesis and enzymes of nitrogen assimilation in greening maize leaf segments: Role of 2-oxoglutarate. Indian J. Exp. Biol. 45, 385–389. Jiang, H.M., Yang, J.C., Zhang, J.F., 2007. Effects of external phosphorus on the cell ultrastructure and the chlorophyll content of maize under cadmium and zinc stress. Environ. Pollut. 147, 750–756. Li, M. S., 2006. Ecological restoration of mineland with particular reference to the metalliferous mine wasteland in China: A review of research and practice. Sci. Total Environ. 357, 38–53. Liu, J., Zhang, X.H., Li, T.Y., Wu, Q.X., Jin Z.J., 2014. Soil characteristics and heavy metal accumulation by native plants in a Mn mining area of Guangxi, South China. Environ. Monit. Assess. 186(4), 2269–2279. Liu, M., Sun, J., Li, Y., Xiao, Y., 2017. Nitrogen fertilizer enhances growth and nutrient uptake of Medicago sativa inoculated with Glomus tortuosum grown in Cd-contaminated acidic soil. Chemosphere 167, 204–211. Luo, J., Li, H., Liu, T., Polle, A., Peng, C., Luo, Z.B., 2013. Nitrogen metabolism of two contrasting poplar species during acclimation to limiting nitrogen nitrogen availability. J. Exp. Bot. 64 (14), 4207–4224.

447

Luo, Z.B., Janz, D., Jiang, X., Go, C., Rennenberg, H., Feussner, I., Polle, A., 2009. Upgrading root physiology for

448

stress tolerance by ectomycorrhizas: insights from metabolite and transcriptional profiling into reprogramming for

449

stress anticipation. Plant Physiol. 151,1902–1917.

450 451

Ma, C., Chen, Y., Ding, S., Li, Z., Shi, W.G., Zhang, Y., Luo, Z.B., 2018. Sulfur nutrition stimulates lead accumulation and alleviates its toxicity in Populus deltoides. Tree Physiol. 38(11), 1724–1741.

452

Ma, Y., He, J., Ma, C., Luo, J., Li, H., Liu, T., Polle A, Peng C.h., Luo, Z.B., 2013. Ectomycorrhizas with Paxillus

453

involutus enhance cadmium uptake and tolerance in Populus × canescens. Plant Cell Environ. 37(3), 627–642.

454

McGrath, S.P., Lombi, E., Gray, C.W., Caille, N., Dunham, S.J., Zhao, F.J., 2006. Field evaluation of Cd and Zn

455

phytoextraction potential by the hyperaccumulators Thlaspi caerulescens and Arabidopsis halleri. Environ. Pollut.

456

141,115–125.

457

Nazar, R., Iqbal, N., Syeed, S., Khan, N.A., 2011. Salicylic acid alleviates decreases in photosynthesis under salt stress

458

by enhancing nitrogen and sulfur assimilation and antioxidant metabolism differentially in two mungbean

459

cultivars. J. Plant Physiol. 168,807–815.

20

460

Qin, Q., Li, X., Wu, H., Zhang, Y., Feng, Q., Tai, P., 2013. Characterization of cadmium (108Cd) distribution and

461

accumulation in Tageteserecta L. seedlings: Effect of split-root and of remove-xylem/phloem. Chemosphere 93,

462

2284–2288.

463 464 465 466 467 468 469 470

Redovniković, I.R., Marco, A.D., Proietti, C., Hanousek, K., Sedak, M., Bilandžić, N., Jakovljević, T., 2017. Poplar response to cadmium and lead soil contamination. Ecotox. Environ. Safe. 144, 482–489. Rees, F., Sterckeman, T., More, J.L., 2016. Root development of non-accumulating and hyperaccumulating plants in metal-contaminated soils amended with biochar. Chemosphere 142, 48–55. Rennenberg, H., Herschbach, C., 2013. Phosphorus nutrition of woody plants: many questions – few answers. Plant Biol. 15, 785–788. Seth, C.S., Remans, T., Keunen, E., Jozefczak, M., Gielen, H., Opdenakker, K., Weyens, N., Vangronsveld, J., Cuypers, A., 2012. Phytoextraction of toxic metals: a central role for glutathione. Plant Cell Environ. 35,334–346.

471

Shi, H., Ma, W., Song, J., Lu, M., Rahman, S.U., Bui, T.T.X., Vu, D.D., Zheng, H., Wang, J., Zhang, Y., 2017.

472

Physiological and transcriptional responses of Catalpa bungei to drought stress under sufficient- and deficient-

473

nitrogen conditions. Tree Physiol. 37 (11),1457–1468.

474 475

Shi, W.G., Li, H., Liu, T.X., Polle, A., Peng, C.H., Luo, Z.B., 2015. Exogenous abscisic acid alleviates zinc uptake and accumulation in Populus × canescens exposed to excess zinc. Plant Cell Environ. 38 (1), 207–223.

476

Sofo, A., Vitti, A., Nuzzaci, M., Tataranni, G., Scopa, A., Vangronsveld, J., Remans, T., Falasca, G., Altamura, M.M.,

477

Degola, F., Sanità di Toppi, L., 2013. Correlation between hormonal homeostasis and morphogenic responses in

478

Arabidopsis thaliana seedlings growing in a Cd/Cu/Zn multi-pollution context. Physiol. Plantarum 149 (4), 487–

479

498.

480

Song, J., Wang, Y., Pan, Y., Pang, J., Zhang, X., Fan, J., Zhang, Y., 2019. The influence of nitrogen availability on

481

anatomical and physiological responses of Populus alba × P. glandulosa to drought stress. BMC. Plant Biol. 19,

482

63.

483 484

Thomas, D.S., Montagu, K.D., Conroy, J.P., 2006. Why does phosphorus limitation increase wood density in Eucalyptus grandis seedlings? Tree Physiol. 26, 35-42.

21

485 486 487 488

Ullah, S., Anwar, S., Rehman, M., 2017. Interactive effect of gibberellic acid and NPK fertilizer combinations on ramie yield and bast fibre quality. Sci. Rep. 7,10647. Vromman, D., Martínez, J.P., Lutts, S., 2017. Phosphorus deficiency modifies As translocation in the halophyte plant species, Atriplex atacamensis. Ecotox. Environ. Safe. 139,344-351.

489

Wang, JC., Wang, H., Chen, J., Zhang, SM., Xu, JT., Han, XT., Feng, YX., Chen, YJ., Zhang, X., Dong, GQ., Zhang,

490

Y., 2019. Xylem development, cadmium bioconcentration, and antioxidant defense in Populus×euramericana

491

stems under combined conditions of nitrogen and cadmium. Environ. Exp. Bot. 164, 1–9.

492

Wu, F.Z., Yang, W.Q., Zhang, J., Zhou, L.Q., 2010. Cadmium accumulation and growth responses of a poplar

493

(Populus deltoids×Populus nigra) in cadmium contaminated purple soil and alluvial soil. J. Hazard. Mater. 177,

494

268–273.

495 496 497 498 499 500

Wu, M., Luo, Q., Liu, S., Zhao, Y., Long, Y., Pan, Y., 2018. Screening ornamental plants to identify potential Cd hyperaccumulators for bioremediation. Ecotox. Environ. Safe. 162, 482–489. Wu, T., Dong, Y., Yu, M., Wang, G.G., Zeng, D.H., 2012. Leaf nitrogen and phosphorus stoichiometry of Quercus species across China. Forest Ecol. Manag. 284,116–123. Yan, B., Ji Z.H., Fan, B., Wang, X.M., He, G.X., Shi, L.T., Liu, G.C., 2016. Plants adapted to nutrient limitation allocate less biomass into stems in an arid-hot grassland. New Phytol. 211(4), 1232–1240.

501

Zhang, S.M., Yang, C., Chen, M.M., Chen, J., Pan, Y.H., Chen, Y.L., Rahman, S., Fan, J.F., Zhang, Y., 2019. Influence

502

of nitrogen availability on Cd accumulation and acclimation strategy of Populus leaves under Cd exposure. Ecotox.

503

Environ. Saf. 180, 439–448.

504 505 506 507

Zheng, H., Zhang, X., Ma, W., Song, J., Rahman, S.U., Wang, J., Zhang, Y., 2017. Morphological and physiological responses to cyclic drought in two contrasting genotypes of Catalpa bungei. Environ Exp. Bot. 138,77–87. Zhu, X.F., Lei, G.J., Jiang, T., Liu, Y., Li G.X., Zheng, S.J., 2012. Cell wall polysaccharides are involved in Pdeficiency-induced Cd exclusion in Arabidopsis thaliana. Planta 236, 989–997.

508 509

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1

Figure legends

2 3 4 5 6

Fig. 1 Plant biomass (a), R/S (b), root biomass (c) and stem biomass (d) of Populus euramericana under combined phosphorus and cadmium treatments. P-Suf, P-sufficient condition (0.6 mM KH2PO4, 0.042 mM K2HPO4); P-Def, P-deficient condition with 10% of phosphorus addition in the P-sufficient condition (0.06 mM KH2PO4, 0.0042 mM K2HPO4); Cd-, no added Cd; Cd+, 4.0 mg kg-1; Cd++, 20 mg kg-1; The bars indicate means

7 8 9

± SE (n = 12). Different letters on the bars indicate significant differences in ANOVA (P < 0.05). P values from two-way ANOVA on phosphorus (P), cadmium (Cd) and their interaction (P × Cd) are indicated (*, P < 0.05; **, P < 0.01; ns, not significant) above each panel.

10 11 12 13 14

Fig. 2 Cadmium localization (a-h) and anatomical properties (i-k) in stems of Populus euramericana under combined treatments of P and Cd. (a) and (e), P sufficient condition and zero Cd exposure (0 mg kg-1). (b) and (f), P sufficient condition and high Cd exposure (20 mg kg-1). (c) and (g), P deficient condition and zero Cd exposure (0 mg kg-1). (d) and (h), P deficient condition and high Cd exposure (20 mg kg-1) Blue bars=200µm. Red

15 16

bars=50µm. Red arrows point to Cd dithizone precipitate. epi, epidermis; col, collenchyma; cor, cortex; ph, pholem; pf, phloem fiber; xyl, xylem.

17 18 19 20

Fig. 3 Cadmium concentration, content and bioconcentration factor in stems. P-Suf, P-sufficient condition (0.6 mM KH2PO4, 0.042 mM K2HPO4); P-Def, P-deficient condition with 10% of phosphorus addition in the Psufficient condition (0.06 mM KH2PO4, 0.0042 mM K2HPO4); Cd-, no added Cd; Cd +, 4 mg kg-1; Cd++, 20 mg

21 22 23

kg-1; The bars indicate means ± SE (n = 4). Different letters above the bars indicate significant differences from ANOVA (P < 0.05). P-values from two-way ANOVA on phosphorus (P), cadmium (Cd) and their interaction (N × Cd) are indicated (*, P < 0.05; **, P < 0.01; ns, not significant) above each panel.

24 25 26 27

Fig. 4 Activity of antioxidant enzymes in stems. APX, ascorbate peroxidase. GR, glutathione reductase. CAT, catalase. POD, peroxidase. P-Suf, P-sufficient condition (0.6 mM KH2PO4, 0.042 mM K2HPO4); P-Def, Pdeficient condition with 10% of phosphorus addition in the P-sufficient condition (0.06 mM KH2PO4, 0.0042 mM

28 29 30 31

K2HPO4); Cd-, no added Cd; Cd +, 4 mg kg-1; Cd ++, 20 mg kg-1; The bars indicate means ± SE (n = 4). Different letters above the bars indicate significant differences by ANOVA (P < 0.05). P-values of two-way ANOVA on phosphorus (P), cadmium (Cd) and their interaction (N × Cd) are indicated above each panel (*, P < 0.05; **, P < 0.01; ns, not significant).

32 33 34 35

Fig. 5 Concentrations of phytohormones in stems. ABA, abscisic acid. JA, jasmonic acid. SA, salicylic acid. GA, gibberellic acid. P-Suf, P-sufficient condition (0.6 mM KH2PO4, 0.042 mM K2HPO4); P-Def, P-deficient condition with 10% of phosphorus addition in the P-sufficient condition (0.06 mM KH2PO4, 0.0042 mM 1

36 37 38 39

K2HPO4); Cd-, no added Cd; Cd+, 4 mg kg-1; Cd++, 20 mg kg-1; The bars indicate means ± SE (n = 4). Different letters above the bars indicate significant differences by ANOVA (P < 0.05). P-values of two-way ANOVA on phosphorus (P), cadmium (Cd) and their interactions (N × Cd) are indicated above each panel (*, P < 0.05; **, P < 0.01; ns, not significant).

40 41 42 43 44 45 46

Fig. 6 PCA of growth and physiological traits under combined conditions of phosphorus and cadmium. Growth traits and physiological parameters including Cd concentrations and content, phosphorus concentrations, nitrogen concentration, N:P stoichiometric ratio, the levels of soluble sugars, free proline, chlorophyll, phytohormones and MDA, and the activities of antioxidant enzymes are included in PCA. Square, P-sufficient condition; Circle, Pdeficient condition. Open circle or square, 0 mg Cd kg−1 sand (CK); Semi-open circle or square, 4 mg Cd kg−1 sand (moderate Cd level); Closed circle or square, 20 mg Cd kg−1 sand (High Cd level).

47 48

Fig. 7 Schematic model of mechanisms underlying the influence of phosphorus status and N:P stoichiometric

49

ratio on cadmium accumulation and stress tolerance in Populus stems. Under P-sufficient condition, cell

50

division and xylem construction were up-regulated by proper tissue P concentration and balanced N:P ratio, which enhanced Cd translocation and increased Cd accumulation in stems. In contrast, the extremely low tissue P concentration and imbalanced N:P ratio under P deficiency reduced cell division and inhibited xylem development, and thus decreased Cd accumulation in stems. Phosphorus addition also increased cell wall affinity for Cd and thus enhanced Cd accumulation in stem cell wall, presumably due to the active polysaccharide production after P application. The activity of APX was down-regulated by low P concentrations and imbalanced N:P ratio under P deficiency, which increased the oxidative stress in stem. A negative interaction between phytohormones and antioxidant enzymes was detected in poplar stems under combined conditions of P deficiency and Cd exposure. In contrast, the positive interactions between phytohormones and antioxidants contributed to the favoring antioxidant defense under normal P conditions. In conclusions, proper P addition and balanced N:P ratio can enhance phytoextraction of Cd from Cd-contaminated sand via enhancing secondary xylem development, up-regulating antioxidant defense and promoting cadmium accumulation in Populus stems.

51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

2

Table 1 Tissue concentrations of P, N and N : P ratio in stems. Phosphorus

N (mg g-1)

N : P Ratio

2.0±0.2 a

9.5±1.9 a

4.5±0.7 b

1.8±0.1 a

8.5±0.6 a

4.9±0.4 b

1.9±0.2 a

8.7±1.2 a

4.7±0.4 b

Cd 0 mg kg-1

0.3±0.04 b

7.3±0.6 a

23.1±3.2 a

-1

0.4±0.03 b

7.8±0.5 a

21.5±1.1 a

Cd 20 mg kg-1

0.4±0.04 b

7.5±0.8 a

22.4±1.8 a

Phosphorus (P)

202.8**

2.7

179.1**

Cadmium (Cd)

0.3

0.04

0.07

P×Cd

0.4

0.3

0.2

Cadmium

P

Cd 0 mg kg-1 Sufficient-P

Cd 4 mg kg

-1

Cd 20 mg kg

Deficient-P

F Value of Two-way ANOVA

Cd 4 mg kg

-1

(mg g-1)

The values are means ± SE (n = 9). Means with different letters are statistically different in one-way ANOVA (P < 0.05). F Value of two-way ANOVA (phosphorus and cadmium) are indicated (*, P < 0.05; **, P < 0.01).

Root Biomass(g)

c

Tree Heig(cm)

e

20

a b

b

b c

c

8 6 4

150 120 90

Cd-

Cd+

0.0

Cd++

d

P ns Cd ns P ×Cd

a a

a

a

a a

Cd-

Cd+

Cd++

P ** Cd ns P ×Cd ns a a a b

b

b

60 30 0

c

d

bc

ab

a c

P-Suf P-Def

0.1

2 0

0.3 0.2

10 0

P ** Cd ** P ×Cd ns

0.4

R/S

30

b 0.5

P ** Cd ** P ×Cd ns

Stem Biomass(g)

Plant Biomass (g)

40

Cd-

Cd+

f

15

Cd-

Cd+

bc

b

12

Cd++

Fig. 1

9

Cd-

P-Suf P-Def

b cd

5 0

Cd++

P ** Cd ** P ×Cd ns a

10

Diameter(mm)

a

Cd+

d

Cd++

P ** Cd ns P ×Cd ns a

P-Suf b

6

a

a b

b

3 0

Cd-

Cd+

Cd++

P-Def

Cd 0 mg kg-1

a

epi col cor

pf ph

Cd 20 mg kg-1

b

epi

col cor

pf ph

P-Suf

xyl

xyl

400µm

c

pf

d

epi cor

P-Def

ph

col

ph

xyl

col

400µm

epi

pf

xyl cor 400µm

400µm

e epi

epi

f

col

col P-Suf

col

P-Def

col cor

1.20

P ** Cd ** P×Cd ns a

1.00

b

0.80

b

0.60

c

0.40 0.20 0.00

Cd -

Cd ++

j Cell Layers of Xylem

Xylem Thickness (mm)

i

50µm

h epi

cor

50µm

k

P ** Cd ** P×Cd ns

50.0

a

40.0

b

30.0

Fiber Cell Diameter

g epi

cor

50µm

cor

b c

20.0 10.0 0.0

Cd -

Cd ++

30.0

50µm P ** Cd ** P×Cd ns a

a

a

a

P-Suf P-Def

20.0 10.0 0.0

Cd -

Cd ++

Fig. 2

a

20 b

15 10 5 0

200

d

b c

d

Cd-

P ** Cd ** P × Cd ns

160

a

120

b

80 40 0

Cd+ Cd++

Fig. 3

4.0

a

BCF

25

Cd content (μg)

Cd concentration (μg g -1 DW)

Stem P ** Cd ** P ×Cd ns

cd

d

Cd-

c

Cd+ Cd++

P * Cd ** P ×Cd * a

P-Suf P-Def

3.0 2.0 1.0 0.0

b

Cd+

b

c

Cd++

9000

a

a

a

a

b b

6000 3000

P * Cd ns P×Cd ns

4000

a

3200 2400

b

1600

P-Suf P-Def

a

ab

ab b

800 0

0 P ** Cd ns P×Cd ns

400

CAT (nakt g-1 prot)

GR (nkat g-1 prot)

P ns Cd ns P×Cd *

12000

300

abc

200

bc

bc

POD (nkat g-1 prot)

APX (nkat g-1 prot)

Stem

a

ab c

100 0

Cd-

Cd+

Cd++

P ns Cd ** P×Cd ns

15000 12000

a ab

ab

9000

P-Def

bc

bc

6000

P-Suf

c

3000 0

Cd-

Cd+

Cd++

P nsStem Cd * P×Cd *

400

a

300 200

JA (ng g-1 DW)

500

b

b

b

b

b

100 0 80

Pns Cd ns P×Cd ** a

60 40 20 0

b

Cd-

a

SA (ng g-1 DW)

GA3(ng g-1 DW)

ABA (ng g-1 DW)

Fig. 4

a b

b

Cd+ Cd++

Fig. 5

P * Cd ** P×Cd *

250 200 150 100 50 0

a a

a

b

6000

0

b

P ns Cd ns P×Cd ** a

4000 2000

b

c

ab bc

P-Suf P-Def

a c

Cd- Cd+ Cd++

P-Suf P-Def

Fig. 6

NADP+

Balanced N:P Ratio

Up

GSH

GR

H2O2

APX

GPX

NADPH

N

P

SOD

O2-•

GSSG

CAT Down

O2+H2O

Imbalanced N:P Ratio P

Free Proline

P P P

Normal P concentration

Down

Up Oxidant Stress

Down Phosphorus Sufficiency Up

P-stimulated cell division and xylem development

Up

N

Low P concentration

Up JA

ABA Cross

GA3 More Cd accumulation, located in cell walls of collenchyma

Fig. 7

Talk

Down

P

Phosphorus Deficiency

SA Less Cd accumulation, located in symplast of collenchyma cells

P

Down

Less cell division and xylem development

HIGH LIGHTS


Sufficient tissue P concentration and balanced N:P ratio enhanced cell division in stem.




Low tissue P concentration and imbalanced N:P ratio inhibited xylem development.




Phosphorus increased cell wall affinity for Cd and Cd accumulation in cell wall in stem.




P deficiency decreased phytohormone levels and increased oxidative stress in stem.




Phosphorus enhanced Cd phytoextraction via promoting xylem development and antioxidant defense.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: