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Zinc Isotope Fractionation in the Hyperaccumulator Noccaea caerulescens and the Nonaccumulating Plant Thlaspi arvense at Low and High Zn Supply Ye-Tao TANG, Christophe Cloquet, Teng-Hao-Bo Deng, Thibault Sterckeman, Guillaume Echevarria, Wen-Jun Yang, Jean-Louis Morel, and Rong-Liang Qiu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b00167 • Publication Date (Web): 30 Jun 2016 Downloaded from http://pubs.acs.org on June 30, 2016

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Environmental Science & Technology

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Zinc Isotope Fractionation in the Hyperaccumulator Noccaea caerulescens and the

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Nonaccumulating Plant Thlaspi arvense at Low and High Zn Supply

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Ye-Tao Tang1,2, Christophe Cloquet3, Teng-Hao-Bo Deng1, Thibault Sterckeman4, Guillaume

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Echevarria4, Wen-Jun Yang1, Jean-Louis Morel4, Rong-Liang Qiu1,2*

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1. School of Environmental Science and Engineering, Sun Yat-sen University, Guangzhou 510275,

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P. R. China

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2. Guangdong Provincial Key Laboratory of Environmental Pollution Control and Remediation

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Technology (Sun Yat-sen University), Guangzhou 510275, P. R. China

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3. CRPG-CNRS, Université de Lorraine (UMR 7358N), 15 rue Notre-Dame-des-Pauvres BP 20,

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54501 Vandoeuvre lès Nancy, France

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4. Laboratoire Sols et Environnement, INRA-Université de Lorraine, 2 avenue de la Forêt de Haye,

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TSA 40602, F-54518 Vandoeuvre-lès-Nancy Cédex, France

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* Corresponding author. Email: [email protected]; Phone number: +86 20 84113454

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ACS Paragon Plus Environment


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Abstract

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Based on our previous field survey, we postulate that the pattern and degree of zinc (Zn) isotope

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fractionation in the Zn hyperaccumulator Noccaea caerulescens (J. & C. Presl) F. K. Mey may

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reflect a relationship between Zn bioavailability and plant uptake strategies. Here, we investigated

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Zn isotope discrimination during Zn uptake and translocation in N. caerulescens and in a

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nonaccumulator Thlaspi arvense L. with a contrasting Zn accumulation ability in response to low

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(Zn-L) and high (Zn-H) Zn supplies. The average isotope fractionation of the N. caerulescens plant

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as a whole, relative to solution (∆66Znplant-solution) were –0.06 and –0.12‰ at Zn-L-C and Zn-H-C,

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respectively, indicative of the predominance of a high-affinity (e.g. ZIP transporter proteins)

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transport across the root cell membrane. For T. arvense, plants were more enriched in light isotopes

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under Zn-H-A (∆66Znplant-solution = –0.26‰) than under Zn-L-A and N. caerulescens plants, implying

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that a low-affinity (e.g. ion channel) transport might begin to function in the nonaccumulating

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plants when external Zn supply increases. Within the root tissues of both species, the apoplast

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fractions retained up to 30% of Zn mass under Zn-H. Moreover, the highest δ66Zn (0.75-0.86‰)

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was found in tightly-bound apoplastic Zn, pointing to the strong sequestration in roots (e.g. binding

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to high-affinity ligands/ precipitation with phosphate) when plants suffer from high Zn stress.

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During translocation, the magnitude of isotope fractionation was significantly greater at Zn-H

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(∆66Znroot–shoot =0.79‰) than at Zn-L, indicating that fractionation mechanisms associated with

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root-shoot translocation might be identical to the two plant species. Hence we clearly demonstrated

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that Zn isotope fractionation could provide insight into the internal sequestration mechanisms of

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roots when plants respond to low and high Zn supplies. 3



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Keywords: isotope fractionation; MC-ICPMS; Noccaea caerulescens; Thlaspi arvense; plant

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physiology; zinc

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Introduction

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Zinc (Zn) is a transition trace element of particular interest, because both Zn deficiency and

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pollution are worldwide issues. As a micronutrient and a cofactor of over 300 enzymes, Zn is

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essential for almost all living organisms, although it can be toxic at elevated levels.1 Soils with low

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Zn concentrations are widespread in the world. It has been reported that 30% of cultivated soils

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globally are deficient in Zn.2 On the other hand, the expansion of industrial and agricultural

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activities (e.g. mining, smeltering, use of fertilizers and pesticides) has led to increasingly severe Zn

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pollution that poses a potential risk to crops and public health.1

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Understanding the mechanisms of Zn absorption, transport and sequestration in plants is a

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premise for both the biofortification of crops and the phytoremediation of contaminated soils.3

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These mechanisms however, are far from being fully understood. In the field of soil

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phytoremediation, intensive studies have focused on a special group of plant species termed Zn

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hyperaccumulators, among which Noccaea caerulescens (J. & C. Presl) F. K. Mey (formerly

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Thlaspi caerulescens) and Arabidopsis halleri (L.) O’Kane and Al-Shehbaz, are regarded as model

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plants for exploring Zn tolerance and hyperaccumulation mechanisms.4-5 The Zn hyperaccumulator

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N. caerulescens is capable of accumulating up to 3% (dry weight) of Zn in its shoots without

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showing any obvious toxicity symptoms.6-7 Compared to the nonaccumulating plant Thlaspi

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arvense L., N. caerulescens presents approximately six-time higher rates of root Zn uptake, which 4


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are assumed to be associated with a greater expression of high-affinity transporters from the ZIP

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(zinc/iron-regulated transporter protein) family.8-9 Much less Zn is sequestered in the root vacuoles

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of N. caerulescens than in T. arvense, in accordance with the hypothesis that the hyperaccumulator

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maintains the root Zn in a more mobile pool that is more readily transferred to xylem.4, 10 Indeed, a

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five-fold higher Zn concentration was found in the xylem sap of N. caerulescens than in that of T.

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arvense, which is presumably driven by an upregulation of heavy-metal-transporting ATPase

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(HMA4).11-12 With regard to Zn chelation and storage in N. caerulescens, it was found that a

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significant fraction of root Zn is coordinated with histidine and the rest is bound to cell walls,13

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while shoot Zn is mostly associated with organic acids in vacuoles of epidermis.13-14

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Recent studies have suggested that the stable Zn isotope fractionation in higher plants could be

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a useful tool for tracing the physiological processes involved in Zn homeostasis.15-29 Weiss and

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co-workers15 have shown for the first time that higher plants discriminate between Zn isotopes.

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They found an enrichment in light Zn isotopes in the shoots, reflecting a biological,

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membrane-transport mediated uptake into plant cells.15 However, rice (Oryza sativa L.) grown in

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paddy soil tends to be enriched in heavy isotopes relative to plant-available Zn, pointing to a key

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role of phytosiderophores (PS) in Zn mobilization and the uptake of Zn-PS by roots.19 The effect of

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root-induced mobilization is further evidenced by Zn isotope fractionation in a Zn-tolerant species

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Agrostis capillaris L. in the presence of Zn-rich technosols25 and in tomato (Lycopersicon

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esculentum L. var. Saint-Pierre) plants exposed to both Zn-deficient and Zn-sufficient conditions.24

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The latter study found a difference of +0.27‰ in ∆66Znplant-solution between low and high Zn supply,

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indicating that Zn-deficiency induced root-exudate complexes participate in the process of Zn 5



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uptake.24 During root uptake, a part of Zn is absorbed on root apoplast (e.g. cell wall), which is

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believed to favor heavy isotope enrichment in roots.15 In fact, there have been several studies

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showing a wide variation (up to 0.8‰) in positive δ66Znroot in a number of plants,15, 20, 22-24 but the

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underlying mechanisms remain elusive. Zinc uptake into root symplast is thought to be mediated by

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a high-affinity and/or a low-affinity transport system. High-affinity transport, i.e. ZIP family

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transporters, would appear to favor the heavy isotope, whereas low-affinity transport, e.g.

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ion-channel and/or electrogenic pumps favors the light one.15, 22 Partly in line with this, the isotopic

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effect in the marine diatom Thalassiosira oceanica (∆66Zncell-media) has been reported as –0.2‰ for

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the high-affinity uptake at low Zn concentrations and –0.8‰ at the highest Zn concentrations,

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where low-affinity uptake is dominant.30 Zn isotopes could further fractionate in favor of light

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isotopes during long-distance root to shoot transport. Moynier et al.18 proposed that a diffusional

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fractionation might dominate Zn transport in xylem, whilst Aucour et al.20 suggested that

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root-to-shoot fractionation is associated with root sequestration in two Arabidopsis species.

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Regarding the Zn sequestration process, Caldelas et al.21 found that isotope fractionation differed

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substantially in a Zn-tolerant species Phragmites australis (Cav.) Trin. ex Steud exposed to

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contrasting Zn levels in solution. The increase in heavier isotopes in roots and lighter isotopes in

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shoots with increasing Zn concentration, is attributed to the more efficient complexation and

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compartmentation of Zn in roots, which protect the plant from toxicity. Together, the above studies

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indicate that the extent of Zn isotope fractionation could be regulated by several factors (e.g. plant

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species, external Zn supplies, plant Zn requirement), although the relationship between isotope

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fractionation and the underlying physiological mechanisms is still unclear. 6


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In a previous field study, we have reported a wide variation in Zn isotope fractionation as

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illustrated by a difference of 0.64‰ between the highest and lowest ∆66Znplant-soil value in N.

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caerulescens collected from three localities with contrasting soil Zn content.23 This allows us to

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hypothesize that the magnitude of Zn isotope fractionation in plants may distinguish between Zn

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uptake strategies and reflect the Zn status in the substrate. More specifically, we propose that when

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external Zn supply changes from low to high level, this would favor an enrichment in lighter

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isotopes in whole plants relative to solution, which may reflect a shift from a high- to a low-affinity

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uptake pathway. Furthermore, using a protocol for separating different fractions in roots, we tried

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for the first time to analyze Zn isotope compositions in the apoplast and symplast root fractions, and

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attempted to link the fractionation with mechanisms for Zn sequestration in root tissues, particularly

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under high Zn stress. Therefore, the objectives of this study were firstly to characterize the Zn

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isotope fractionation associated with Zn uptake and translocation in the Zn hyperaccumulator N.

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caerulescens grown in hydroponics under low and high Zn conditions in comparison with the

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nonaccumulating species T. arvense; secondly, to demonstrate the contribution of root apoplast and

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symplast to root Zn uptake and isotope fractionation within roots in response to low and high Zn

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supply; thirdly, to quantify the magnitude of Zn isotope fractionation associated with root-to-shoot

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translocation in the two plants tested.

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Materials and Methods

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Plant growth and Zn treatment

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The details of plant germination and cultivation are given in the Supporting Information (M&M 7



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section). Due to the faster growth rate of T. arvense compared to that of N. caerulescens, thirty-five

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days (for N. caerulescens) and fifteen days (for T. arvense) after transfer to solution, seedlings were

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treated with low and high Zn (respectively 1 µM (Zn-L-C) and 50 µM (Zn-H-C) for N.

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caerulescens; 0.02 µM (Zn-L-A) and 5 µM (Zn-H-A) for T. arvense) in a plastic tray containing the

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same nutrient concentrations as the agar germination medium. Each plant was treated as a single

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replicate, and there were three replicates of each. The solution was renewed weekly and deionized

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water was added daily to compensate for the loss of water due to evaporation and transpiration. For

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Zn-L and Zn-H treatment, the whole exposure duration was 14 d for T. arvense and 28 d for N.

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caerulescens. Then three plants of each species were harvested and separated into shoots and roots

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before tissue Zn extraction and determination procedures.

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Separation and treatment of plant tissues

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Following harvesting, root Zn was separated into three fractions. Firstly, the exchangeable

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apoplastic fraction (F1), where the roots were desorbed by 80 mL 5 mM CaCl2 solution (pH 5.7

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buffered with 2 mM MES) in an ice-bath (0°C) for 60 min. Secondly, the symplastic fraction (F2),

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where after desorption, roots were immersed in 120 mL of a methanol–chloroform mixture (2/1,

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v/v) for 3 d.8, 31 This treatment dissolves the cytoplasmic membrane without modifying the cell wall

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composition. The roots were then transferred into two successive baths of distilled water (120 mL

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and 60 mL), in which they were left to stand for 24 h each. Vigorous manual shaking was

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performed twice a day, to extract intracellular compounds from the roots. Root tissues were finally

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immersed again in 60 mL of a desorption solution (5 mM CaCl2, pH 5.7 buffered with 2 mM MES)

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for 60 min at 0 °C, in order to desorb some cytoplastic Zn which may have adsorbed onto the cell 8


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walls after cell lyses. The symplast fraction was a combination of three extractions from methanol–

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chloroform, deionized water and CaCl2. The third fraction (F3) was non-exchangeable apoplast

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from the roots remaining after symplast separation. The desorbed solutions and symplast extracts

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were filtered at 0.20 µm and stored at 4 °C until chemical analysis.

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After harvesting, the shoots were carefully rinsed three times with deionized water and blotted

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dry. Then shoot samples and the non-exchangeable root fractions (F3) were dried at 60 °C to a

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constant weight, and ground into a fine powder (0.5 mm sieve). The biomass of plant samples was

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measured on a dry weight basis.

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Analytical methods

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To measure Zn concentration in dry plant tissues (leaf and nonexchangeable root, F3), test

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portions (100 mg) were digested in concentrated HNO3-H2O2-HCl (sub-boiled distilled, analytical

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grade) mixtures using a High Pressure Asher (HPA-S, Anton Paar, Austria) at 300°C for 180 min.

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All digests and an aliquot of 10-50 mL root extract samples (F1 and F2 fractions) were evaporated

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to dryness, and the residues were dissolved in 1 mL of 6 M HCl. The resulting solutions were split

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into two parts: 0.1 mL for Zn concentration measurement by ICP-MS (Perkin-Elmer ICP-MS

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SCIEX Elan 6000 or Thermo X7 located at CRPG, Nancy, France), and 0.9 mL for isotope analysis.

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Water used for dissolution and dilution was purified using a Millipore deionizing system at 18.2

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MΩ cm. Other reagents used for sample preparation (HNO3 and HCl) were sub-boiled for isotope

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analyses. During the whole process, blank tests were conducted to estimate the level of

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contamination induced by acid digestion. The average blank measured throughout the study was 35

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± 5 ng of Zn (n=3), which is negligible compared to the Zn contents in samples (0.5-460 µg). The 9



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international reference material (BCR-62, Olea europaea L.) was used to check the validity and the

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reproducibility of both the acid digestion and ICP-MS analysis. The Zn concentration obtained for

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BCR-62 was 16.8 ± 0.6 g g-1 (n=4), which is similar to the previous report.23

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Measurements of Zn isotope analysis are detailed in Cloquet et al.32 and Tang et al.23 Briefly,

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AGMP1 (100-200 mesh, chloride form) anion exchange resin (Bio-Rad laboratories, CA, USA) was

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used to remove matrix components from Zn. Then the Zn fraction was dried and re-dissolved in 1

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mL 0.1 M HNO3 with an ultrasonic bath for 30 min. To avoid artificial isotope fractionation by the

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column chemistry, Zn recovery was quantitatively monitored and yielded a range of 96.0-103.0% in

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the samples. Zinc isotopes were measured with a Neptune MC-ICP-MS (ThermoFinnigan) at CRPG,

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Nancy (France). The standard-sample-standard bracketing technique was applied and the

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instrumental mass bias was corrected according to the empirical method previously published31,

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using Cu NIST 976 as an external normalizing element for Zn. Results obtained are expressed as a

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delta per mill (‰) notation relative to the ZnIRMM-3702 reference solution. ZnJMC Lyon was regularly

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analyzed throughout the study gaining a δ66Zn = –0.28 ± 0.05 ‰ (2 SD, n=27). The BCR-62 was

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also analyzed throughout the measurements, having a δ66Zn value of 0.16 ± 0.05‰ (2 SD, n=4).

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These values are in agreement with those previously published.23, 26, 32

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The average isotopic composition of the whole plant and root was calculated according to the

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following equations:

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66 =

200 201

∑

66

(1)

∑

Where δ66Zni, mi and ci are the Zn isotope composition (‰), biomass (g) and Zn concentration (mg kg-1) of plant part i (root or shoot). 10


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66

∑

66

202

=

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Where δ66Zni and ci are the Zn isotope composition (‰) and Zn concentration (mg kg-1) of each

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root fraction (F1, F2, and F3).

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Statistical analysis

(2)

∑

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SPSS version 22.0 for Windows was used for data analysis. One-way analysis of variance

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(ANOVA) was performed on the plant biomass, Zn isotope compositions in the root fractions and

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shoots, and the Zn isotope fractionation between solution and plant and between root and shoot.

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Logarithmic transformation was performed when data did not meet the assumption of equal

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variances. Turkey’s HSD test was used for multiple comparison at P < 0.05 level between treatment

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means.

212 213

Results

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Zn concentrations in root fractions and shoots

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Overall, the plants grew healthily without any obvious symptoms of Zn deficiency or toxicity

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under all Zn treatments (Figure S1, Supporting Information). In the Zn-L-C, plants of N.

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caerulescens accumulated comparable amount of Zn in the roots and shoots (ca. 0.5 and 0.6 mg g-1)

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with a translocation factor (TF, shoot Zn concentration / root Zn concentration) of 1.34 (Fig. 1).

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However, the TF value decreased to 0.57 at Zn-H-C, although a greater shoot concentration (9.0 mg

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g-1) was obtained. In the plants of T. arvense, Zn concentration was much higher in the roots than in

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the shoots with the TF values ranging from 0.03 to 0.24 in the two Zn treatments.

11



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With regard to individual root fractions, the symplastic fraction (F2) was dominant,

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representing 68% to 93% of the root Zn content, with a maximum of 93% and 88% at Zn-L for N.

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caerulescens and T. arvense, respectively (Table S1, Supporting Information). The apoplastic Zn

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fraction (sum of the exchangeable fraction (F1) and the nonexchangeable fraction (F3)) was

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relatively low (7-32%). It is noticeable that more Zn was adsorbed on apoplast when plants were

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exposed to high Zn levels in solution.

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Zn isotopes in plants

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Zn isotopes in root fractions and shoots. Under Zn-H, all root fractions were consistently

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enriched in heavy isotopes relative to the initial solution, with δ66Zn values being 0.34–0.63‰ for N.

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caerulescens and 0.17–0.75‰ for T. arvense (Fig. 2a). However, the Zn-L treatment led to a slight

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depletion in heavy isotopes relative to initial solution in the exchangeable root fraction (F1) of both

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plants and in the root symplast of T. arvense, with δ66Zn values ranging from –0.30 to –0.10‰.

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Under all Zn treatments except Zn-L-A, the non-exchangeable fractions (F3) had δ66Zn values of

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0.36–0.86‰ for N. caerulescens and 0.53-0.75‰ for T. arvense. Overall, the Zn isotopic signature

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of the symplastic faction (F2) was largely representative of the whole root, because this fraction was

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dominantly present (67–93%) in all Zn treatments (Table S1, Supporting Information). As a result,

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the whole roots were enriched in heavy Zn relative to the initial solution, except in the Zn-L-A

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treated plants of T. arvense.

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Noticeably, the depletion in heavy Zn in shoots was dependent on Zn concentrations in

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solution and showed a species-specific pattern (Fig. 2b). While Zn-H resulted in a greater depletion

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in heavy Zn compared to Zn-L, plants of T. arvense showed consistently more negative δ66Zn

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values relative to N. caerulescens under their corresponding Zn-L or Zn-H treatments.

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Zn isotope fractionation from root to shoot. The greatest isotope fractionation was observed

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during Zn translocation with an identical ∆66Znshoot–root value of –0.79‰ for both species at Zn-H

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(Fig. 3). The Zn isotope fractionation became less pronounced for N. caerulescens at Zn-L-C

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(∆66Znshoot–root= –0.47‰) and for T. arvense at Zn-L-A (∆66Znshoot–root= –0.29‰), indicating that Zn

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supply level had a substantial impact on Zn isotope fractionation during root-shoot translocation.

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Moreover, the Zn isotope composition in roots and shoots of N. caerulescens showed a positive

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shift relative to T. arvense under Zn-L or Zn-H.

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Zn isotope fractionation from solution to whole plant. In all cases, the whole plant tended to be

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enriched in light isotopes relative to the initial solution with ∆66ZnWhole plant– solution values of –0.06

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and –0.12‰ for N. caerulescens and of –0.16 and –0.26‰ for T. arvense at their corresponding

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Zn-L and Zn-H treatments (Fig. 3). The extent of depletion in heavy isotopes in both species was

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more significant at Zn-H than at Zn-L. Furthermore, the Zn isotope composition of the whole plants

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of N. caerulescens was significantly heavier than that in T. arvense under their corresponding Zn-L

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or Zn-H treatments.

258 259

Discussion

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Zinc isotope fractionation during uptake in response to low and high Zn supply

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The Zn supply level has an impact on Zn isotope compositions in the whole plant grown in

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hydroponics. The fractionation between whole plant and solution yielded an overall depletion in 13



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heavy isotopes (Fig. 3). Noticeably, the magnitude of this depletion was more pronounced in the

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Zn-H than in the Zn-L treatment.

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The majority of Zn (68–93%) being in the symplast (F2) root fraction relative to the whole

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roots (Fig. 2a, Table S1, Supporting Information) suggests that the Zn isotopic signatures in the

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whole plant are predominantly governed by transport-mediated uptake. Zinc uptake is thought to be

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metabolically mediated by two different transport systems: a high-affinity transport system i.e. a

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protein carrier, that mainly operates at low Zn conditions, and a low-affinity transport system i.e.

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ion channels and electrogenic pumps, that is dominant under conditions with high Zn

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concentrations.33 High-affinity transport would appear to favor the heavy isotope because of

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covalent binding to a carrier protein i.e.. ZIP, while low-affinity transport seems to prefer light

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isotopes because of their greater diffusion coefficient.15, 22 However, the isotope effect correlated

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with Zn ion binding to proteins, may essentially depend on the nature of the ligands involved at the

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binding sites, as indicated by the recent theoretical work of Fujii et al.34 They suggested that heavy

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isotopes tend to bind to O-donor ligands e.g. aqueous Zn2+ and light isotopes bind to S-donor

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ligands e.g. aqueous Zn(cysteine)2+, while isotope fractionation associated with N-donor ligands

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e.g. aqueous Zn(histidine)2+ may be intermediate between O- and S- donor ligands or even stronger

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than O-donor ligands. So far, no direct evidence links the two transport systems with isotopic

280

discrimination in higher plants. Nevertheless, it has been found that a switch from high-affinity to

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low-affinity transport in marine diatoms resulted in a depletion in heavy isotopes (∆66Zncell-media)

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from −0.2‰ at low Zn to −0.8‰ at high Zn conditions.30 In agreement with this, a slight preference

14


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for light Zn isotopes in the two Arabidopsis species (∆66Znplant-solution= −0.19‰ to −0.05‰) was

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attributed to a high-affinity transport system, i.e. ZIP-transporters.20

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Molecular studies have demonstrated that Zn2+ is taken up by at least six different families of

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transporters, including ZIPs.2 It has been shown that ZNT1, one of the ZIP transporters, is more

287

strongly expressed in the roots of N. caerulescens exposed to Zn-deficiency than in those exposed

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to Zn excess, and the nonaccumulating species T. arvense grown in both Zn-deficiency and

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Zn-excess.9 This observation is consistent with the hypothesis that Zn hyperaccumulation in N.

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caerulescens is caused at least partly, by increased expression of Zn transporters in the root.9, 35

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The effect of high- and low-affinity transport systems on isotope discrimination is critical to

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explaining our experimental results. N. caerulescens, the Zn hyperaccumulator, was slightly

293

enriched in light isotopes relative to the solution at Zn-L-C (−0.06‰) and at Zn-H-C (−0.12‰, Fig.

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3). This matches the result (∆66Znplant-solution= −0.05‰) for the Zn hyperaccumulator A. halleri,20

295

indicating that the ZIP-transporters which function effectively in a wide range of external Zn

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supplies (0–50 µM) in N. caerulescens,9 have a high-affinity for Zn. We postulate that the

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high-affinity transport would not discriminate isotopes and the fractionation observed might be due

298

to a diffusional fractionation which occurs in solution at the root surface. There might be a

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depletion gradient of Zn, even if the solution is stirred.36 In our case, 75–90% of Zn in the Zn-L-C

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solution and 9-91% of Zn in the Zn-H-C solution were depleted within one week during the 4-w

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growth period (Table S2, Supporting Information), indicating a rapid uptake by N. caerulescens

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roots and probably the existence of an ion diffusion zone at the root surface.26 The slight difference

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in ∆66Znplant-solution between Zn-L-C and Zn-H-C might be inferred from the change in Zn isotope 15



304

solution composition (before and after renewal) in the course of experiment (Table S2, Supporting

305

Information). At Zn-L-C, a slight enrichment in δ66Zn (0.02-0.13‰) in the remaining solution was

306

observed during the 4-w growth period. At Zn-H-C, a significant enrichment in δ66Zn (0.28-0.71‰)

307

in the remaining solution was found after 3-4-w growth, indicating that a greater number of light Zn

308

isotopes are absorbed by the plants at Zn-H-C. In line with the study of A. halleri,20 a Rayleigh

309

fractionation model suggests that Zn isotope composition in the solution is a function of the Zn

310

fraction left in solution for N. caerulescens at Zn-H-C (Fig. S2, Supporting Information).

311

For T. arvense, the nonaccumulator, plants were isotopically lighter relative to the solution by

312

–0.16‰ at Zn-L-A and –0.26‰ at Zn-H-A (Fig. 3). Because isotope fractionation linked with Zn

313

uptake transport in higher plants has not been reported on, we can only hypothesize as to its

314

underlying mechanisms. Despite being less pronounced than the marine diatom,30 increasing Zn in

315

solution did significantly enhance the magnitude of discrimination by 62%. It has been reported that

316

10 nM of Zn is a critical concentration for the shift from high- to low-affinity transport in bread

317

wheat.33 In the marine diatom, the critical concentration threshold is even lower.30 In a previous

318

molecular study, a much lower expression of ZNT1 and around a 2-fold decrease in Vmax for Zn

319

influx was found in T. arvense at 1-10 µM Zn treatments than at Zn-deficiency (0 µM)9. This would

320

suggest that the high-affinity transport system at least, became less pronounced in the Zn-H-A

321

treated T. arvense. Thus our results indicate that there might be a trend of switching from high- to

322

low-affinity transport for T. arvense when the external Zn concentration increases. However, the

323

Zn-H-A (5 µM) is probably insufficient to induce the large negative shift observed in diatoms

324

(∆66Zncell-media = −0.8‰).30 It should be noted that the desorption protocol may also have an impact 16


Page 16 of 34

Page 17 of 34


325

on isotopic fractionation during uptake. In the marine diatom experiment, the cell was washed with

326

an oxalate-EDTA agent (50 mM-100 mM),30 which is presumed to be effective for removing

327

extracellular Zn.37 In our case, provided that the exchangeable (F1) and non-exchangeable (F3)

328

fractions of root had been completely removed by the desorption agent, this would lead to a

329

fractionation of −0.34‰ in ∆66Znplant-solution at Zn-H-A for T. arvense, while there was no change at

330

Zn-L-A (∆66Znplant-solution = −0.15‰). It is apparent that when adsorption on root apoplast becomes

331

more significant with the increase of Zn supply, it would more markedly bias the isotopic

332

composition against light Zn in the whole plant.

333

Zn isotope fractionation within roots in response to low and high Zn supply

334

Zinc is primarily taken up by plant roots via two pathways: an apoplastic and a symplastic

335

route. The Zn isotopic compositions in roots should therefore represent the combined effect of

336

sorption to root apoplast, influx via uptake transporters and translocation to shoot. Current root

337

δ66Zn data have mostly been available for the whole roots, which have shown wide variations

338

among plant species. For instance, the roots of rice, tomato and lettuce showed a slight enrichment

339

(0.08–0.18‰) in heavy Zn without significant difference among species.15 However, when the Zn

340

hyperaccumulator A. halleri and the nonaccumulating plant A. petraea were exposed to 10 or 250

341

µM Zn in solution, their root δ66Zn values presented a wide gap of up to 0.8‰ with a preference for

342

heavier isotopes in the former.20 In the Zn-tolerant plant P. australis, root δ66Zn values differed

343

substantially (0.3‰) when seedlings were exposed to Zn-sufficient and Zn-excess conditions.21

344

These results indicate that the overall root isotopic compositions are highly species-specific and can

345

be influenced by the external Zn supply, but the underlying mechanism remains unknown. 17



346

Page 18 of 34

Our experiment distinguished the apoplastic and symplastic roots by using a series of

347

desorption agents that are widely adopted for uptake isotherm studies.8,

348

weakly-bound apoplast fraction (F1), which accounted for only 4-8% of total Zn in roots, was

349

depleted in heavy Zn for N. caerulescens (–0.10‰) and T. arvense (–0.30‰) respectively (Fig. 2a,

350

Table S1, Supporting Information). Because the apoplast is the first compartment in contact with

351

external solution, the lighter isotopes in the F1 fraction should reflect the isotopic composition of

352

the ions initially entering the plant. Indeed, there was no (N. caerulescens) or only slight isotope

353

fractionation (T. arvense, –0.11‰) between the F1 fraction and the whole plants (Table S1,

354

Supporting Information), which supports this hypothesis. In T. arvense’s roots, the symplast

355

fraction (F2) and the tightly-bound apoplast fraction (F3) were also slightly enriched in light

356

isotopes without significantly differing from the whole plant. This is because most Zn entering the

357

plant (78%, Table S1, Supporting Information) was trapped in the root at Zn-L-A, thus the final root

358

δ66Zn should fall close to being the total absorbed Zn. In N. caerulescens however, the largest

359

positive fractionation of 0.96‰ was observed between the F1 and F3 fractions, which is presumably

360

due to a strong covalent bonding to high affinity ligands in cell walls during the radical transport

361

within root apoplast. Despite being less pronounced, Aucour et al.39 reported that in the roots of a

362

wetland plant Phalaris arundinacea, HCl-extracted Zn fraction which may represent Zn strongly

363

bound to cell wall tetrahedral sites, had a heavier δ66ZnLyon (0.32‰) than that of the CaCl2 extracted

364

fraction (0.06‰). The predominance of free carboxyl and hydroxyl groups of pectins in the root cell

365

wall40 might be responsible for the binding,15, 21 but it is still unclear which specific ligands are

366

involved. An alternative explanation is that the F3 fraction is enriched in much heavier isotopes as a 18


31, 38

At Zn-L, the

Page 19 of 34


367

result of interaction of Zn with phosphates in the apoplast. Recently, Fujii and Albarède41 calculated

368

the Zn isotope effect in aqueous citrates, malates and phosphates using ab initio methods. The

369

authors suggested a large enrichment in heavy isotopes of Zn phosphates relative to the free Zn ions

370

at solution pH of 5–7, a pH range that is normal in root tissues. In an earlier study, a strong

371

correlation was observed between the insoluble P and insoluble Zn in roots of N. caerulescens with

372

a P: Zn ratio close to Zn3(PO4)2, when the plants were supplied with a wide range of Zn in solution

373

(1–1000 µM).42 Therefore precipitation of Zn phosphates is likely to occur on the root surface or in

374

the apoplast, explaining the large positive fractionation occurred in the F3 fraction. It should be

375

noted that the Zn concentration in solution was much higher for N. caerulescens (1 µM) than for T.

376

arvense (0.02 µM) at Zn-L, which may also affect Zn precipitation in the apoplast. Besides, the fact

377

that Zn pumping by the root cell in the external medium favors the light isotope, suggests that the

378

enrichment in heavy isotopes of the tightly bound apoplastic Zn might in part be due to the selective

379

pumping of light isotopes at the symplasm membrane within the inner cortex. Once the external Zn

380

ions have entered the cell via a symplast transport system, they would either be chelated with

381

ligands in cytoplasm or be transferred into the vacuoles by metal tolerance proteins (MTPs)

382

localized in the tonoplast, before being further translocated to the shoots.4 Both of the processes are

383

mass-dependent and thus expected to favor the heavy isotopes, leading to an enrichment in heavy

384

isotopes of the symplast root fraction (0.28‰). Although within the framework of this study it is

385

hard to distinguish between the two processes, the positive isotope fractionation observed is

386

consistent with isotopic exchange between the free Zn2+ ion and Zn bound to a high affinity ligand

387

i.e. functional groups of humic acids, in solution.43 19



388

At Zn-H, the isotopic compositions of all the three root fractions differed from those at Zn-L.

389

Firstly, the exchangeable fraction (F1) of both plant species was enriched in heavy isotopes with

390

δ66Zn around 0.5‰ heavier than at Zn-L (Fig. 2a, Table S1, Supporting Information). This might be

391

attributed to a stronger adsorption of Zn (F1, 16%) bound to cell walls which is expected to be

392

enriched in heavier Zn when external Zn supply increases. Secondly, the nonexchangeable root

393

fraction (F3) showed a much heavier δ66Zn value (0.75‰) in T. arvense at Zn-H-A relative to

394

Zn-L-A. This result is similar to that observed in N. caerulescens, indicative of the strongest

395

binding to high-affinity ligands, or a precipitation of Zn phosphates on root apoplast as discussed

396

earlier. Thirdly, as for the symplast fraction (F2), again, T. arvense was preferentially enriched in

397

heavier isotopes at Zn-H-A than at Zn-L-A, while similar δ66Zn values (0.28–0.34‰) were found in

398

N. caerulescens. By using X-ray absorption spectroscopy (XAS), Salt et al.13 reported that for N.

399

caerulescens grown hydroponically with 50 µM Zn, the majority of Zn (70%) in roots is

400

coordinated with histidine with the rest complexed to the cell wall. Because of the high stability

401

constant of the Zn histidine complex at the pH values commonly found in the cytoplasm (pH～7.5),

402

it could be speculated that the positive isotope fractionation observed in the symplast fraction (F2)

403

might result from the complexation of Zn with histidine in the root cytoplasm. Though isotope

404

fractionation linked with the chelation of Zn with histidine has not yet been documented, our results

405

support the hypothesis that the large positive isotope fractionation (～0.4‰) was associated with

406

cellular sequestration in roots.20

407

Zn isotope fractionation from root to shoot in response to low and high Zn

20


Page 20 of 34

Page 21 of 34

408


Under all the Zn treatments, the shoots of both plants were exclusively impoverished in heavy

409

Zn (Fig. 2b), suggesting that root-shoot translocation favors light isotopes.15,

18, 20-23, 25-27

410

particular, the discrimination was significantly greater at Zn-H (0.79‰) than at Zn-L (0.29–0.47‰)

411

regardless of plant species (Fig. 3, Table S1, Supporting Information). Similarly, for the Zn-tolerant

412

species P. australis, the fractionation between the living roots and shoots reached 1.0‰ under

413

Zn-excess, much higher than under Zn-sufficiency.21 Also, tomato seedlings showed ∆66Znroot-shoot

414

values around 0.5‰ greater in the high Zn than in the low Zn treatment.24 Aucour et al.20 found that

415

the average ∆66Znroot-shoot values were similar (–0.66‰ and –0.64‰) for the Zn hyperaccumulator A.

416

halleri and the nonaccumulating A. petraea, and they suggested with a Rayleigh mass-balance

417

model that the mechanisms of root-shoot translocation may be identical in the two Arabidopsis

418

species, with the exception of the Zn fluxes involved. Together, these observations imply that

419

fractionation associated with root-shoot translocation is independent of plant species and would

420

appear to be governed by specific mechanisms in response to varying external Zn conditions.

In

421

Several root sequestration mechanisms (e.g. absorption, precipitation and complexation) are

422

likely to be involved in the pattern observed during translocation under high Zn stress. All these

423

processes are assumed to choose the heavy isotopes, resulting in lighter ones loading into the xylem

424

and the shoots. In our case, approximately 70% of Zn in the Zn-H roots was distributed in symplast,

425

while the rest was bound to the apoplast (Table S1, Supporting Information). As discussed in the

426

previous section, the complexation with histidine combined with the rest of Zn precipitated by

427

phosphate in apoplast, may explain the more positive δ66Zn values in the Zn-H roots and the lighter

428

isotopes being transferred into xylem sap and shoots. 21



429

Environmental implications

430

The present study suggests that Zn supply levels could be an important factor affecting Zn

431

isotope fractionation, which in turn reflects different assimilation, sequestration and translocation

432

mechanisms in plants. Firstly, a high Zn supply would generate a more intense fractionation toward

433

light isotopes in the whole plants relative to solution, than those with a low Zn supply. The

434

fractionation was more pronounced in T. arvense, indicating that low-affinity transport might begin

435

to function in nonaccumulating plants, especially under high Zn supply. In contrast, high-affinity

436

transport, which normally functions in the hyperaccumulator N. caerulescens, would probably not

437

or only slightly fractionate Zn toward light isotopes. These results are somehow contradictory to the

438

conceptual model proposed by Jouvin et al.22, who suggest that uptake by ZIP transporters could

439

lead to a fractionation toward the uptake of heavy isotopes. Thus more studies should be conducted

440

to give a clear profile of the isotope effect associated with Zn-binding to such proteins as ZIP, as

441

illustrated by the theoretical work of Fujii et al.34 Secondly, under high Zn, the root apoplast

442

retained a substantial amount of Zn (ca. 30%) and showed the highest δ66Zn (up to 0.86‰) in

443

tightly-bound apoplastic Zn. This points to the functioning of strong sequestration processes i.e.

444

binding to high-affinity ligands/precipitation with phosphate, when roots suffer from high Zn

445

concentrations. Moreover, the magnitude of isotope fractionation during translocation was much

446

greater at Zn-H than at Zn-L. These observations imply that δ66Zn of roots and root-shoot

447

fractionation might be good indicators for monitoring plant responses to high Zn stress and tracing

448

sequestration mechanisms in roots. Thirdly, those studies with same plant species (i.e. N.

449

caerulescens) allow us to compare Zn absorption mechanisms in the plants grown in both 22


Page 22 of 34

Page 23 of 34


450

hydroponic and field conditions. In a previous field study, we observed a greater enrichment in

451

heavy isotopes of up to 0.63‰ in ∆66Znplant–soil for the serpentine soil grown N. caerulescens.23

452

However, the positive fractionation in whole plants relative to the medium, has rarely been

453

documented in the previous hydroponic experiments.15, 20-22 The great inconsistency between the

454

two conditions indicates that isotope fractionation in natural plant-soil systems is far more

455

complicated than we expected. So far, there is scarce information available, which suggests that

456

enrichment of plants with heavy isotopes is probably due to a phytosiderophore-Zn uptake by rice

457

in Zn-deficient soil,19 or to adsorption of light Zn on Fe/Mn oxides.25 Recently, our field study

458

found that the whole plants of Ni hyperaccumulators grown on serpentine sites were isotopically

459

heavier than the soil (∆60Niplant-soil up to 0.4‰), and fractions of Ni extracted by DTPA were

460

isotopically heavier than the soil (∆60NiDTPA-soil up to 0.89‰).44 This result may provide an insight

461

into isotope fractionation mechanisms associated with uptake of a metal from the bioavailable pool

462

of the soil. We hope to clarify this in future studies.

463 464

Acknowledgements

465

We thank Delphine Yeghicheyan, Aymeric Schumacher (CRPG) for technical support in ICP-MS

466

and MC-ICPMS measurements. This work was financially supported by the Natural Science of

467

China (No. 41371315, No. 41225004), the Fundamental Research Funds for the Central

468

Universities (No.15lgjc36), ANR-10-LABX-21 (LABEX Ressouurces21), and the ANR Arctic

469

Metals (ANR 2011 CESA 011-01). We are grateful to the four anonymous reviewers for their

470

valuable comments and suggestions. 23



471 472

Supporting Information

473

Experimental details and results for plant growth (Figure S1); Zn isotope composition as a function

474

of fraction of Zn left (f) for nutrient solution of N. caerulescens plants in the Zn-H-C treatment

475

(Figure S2); Zn concentrations, isotopic compositions, biomass of roots, shoots and whole plants in

476

the hydroponically grown Noccaea caerulescens and Thlaspi arvense exposed to low and high Zn

477

levels in solution (Figure S1); Zn concentration and δ66Zn in the nutrient solution over time (Table

478

S2). This information is available free of charge via the Internet at http://pubs.acs.org/.

479

References

480

1. Broadley, M. R.; White, P. J.; Hammond, J. P.; Zelko, I.; Lux, A. Zinc in plants. New Phytol.

481

2007, 173 (4), 677–702.

482

2. Hacisalihoglu, G.; Kochian, L. V. How do some plants tolerate low levels of soil zinc?

483

Mechanisms of zinc efficiency in crop plants. New Phytol. 2003, 159 (2), 341–350.

484

3. Zhao, F. J.; McGrath, S. P. Biofortification and phytoremediation. Curr. Opin. Plant Biol.

485

2009, 12 (3), 373–380.

486

4. Milner, M. J.; Kochian, L. V. Investigating heavy-metal hyperaccumulation using Thlaspi

487

caerulescens as a model system. Ann. Bot. 2008, 102 (1), 3–13.

488

5. Roosens, N. H. C. J.; Willems, G.; Saumitou-Laprade, P. Using Arabidopsis to explore zinc

489

tolerance and hyperaccumulation. Trends Plant Sci. 2008, 13 (5), 208–215.

24


Page 24 of 34

Page 25 of 34


490

6. Brown, S. L.; Chaney, R. L.; Angle, J. S.; Baker, A. J. M. Zinc and cadmium uptake by

491

hyperaccumulator Thlaspi caerulescens and metal tolerant Silene vulgaris grown on

492

sludge-amended soils. Environ. Sci. Technol. 1995, 29 (6), 1581–1585.

493

7. Shen, Z. G.; Zhao, F. J.; McGrath, S. P. Uptake and transport of zinc in the hyperaccumulator

494

Thlaspi caerulescens and the non-hyperaccumulator Thlaspi ochroleucum. Plant Cell Environ.

495

1997, 20 (7), 898–906.

496

8. Lasat, M. M.; Baker, A.; Kochian, L. V. Physiological characterization of root Zn2+ absorption

497

and translocation to shoots in Zn hyperaccumulator and nonaccumulator species of Thlaspi. Plant

498

Physiol. 1996, 112 (4), 1715–1722.

499

9. Pence, N. S.; Larsen, P. B.; Ebbs, S. D.; Letham, D. L. D.; Lasat, M. M.; Garvin, D. F.; Eide,

500

D.; Kochian, L. V. The molecular physiology of heavy metal transport in the Zn/Cd

501

hyperaccumulator Thlaspi caerulescens. Proc. Natl. Acad. Sci. U.S.A. 2000, 97 (9), 4956–4960.

502

10. Lasat, M. M.; Baker, A. J. M.; Kochian, L. V. Altered Zn compartmentation in the root

503

symplasm and stimulated Zn absorption into the leaf as mechanisms involved in Zn

504

hyperaccumulation in Thlaspi caerulescens. Plant Physiol. 1998, 118 (3), 875–883.

505

11. Papoyan, A.; Kochian, L. V. Identification of Thlaspi caerulescens genes that may be involved

506

in heavy metal hyperaccumulation and tolerance: characterization of a novel heavy metal

507

transporting ATPase. Plant Physiol. 2004, 136 (3), 3814–3823.

508

12. Hanikenne, M.; Talke, I. N.; Haydon, M. J.; Lanz, C.; Nolte, A.; Motte, P.; Kroymann, J.;

509

Weigel, D.; Krämer, U. Evolution of metal hyperaccumulation required cis-regulatory changes and

510

triplication of HMA4. Nature 2008, 453 (7193), 391–395. 25



511

13. Salt, D. E.; Prince, R. C.; Baker, A. J. M.; Raskin, I.; Pickering, I. J. Zinc ligands in the metal

512

hyperaccumulator Thlaspi caerulescens as determined using x-ray absorption spectroscopy.

513

Environ. Sci. Technol. 1999, 33 (5), 713–717.

514

14. Küpper, H.; Jie Zhao, F.; McGrath, S. P. Cellular compartmentation of zinc in leaves of the

515

hyperaccumulator Thlaspi caerulescens. Plant Physiol. 1999, 119 (1), 305–312.

516

15. Weiss, D. J.; Mason, T. F. D.; Zhao, F. J.; Kirk, G. J. D.; Coles, B. J.; Horstwood, M. S. A.

517

Isotopic discrimination of zinc in higher plants. New Phytol. 2005, 165 (3), 703–710.

518

16. Viers, J.; Oliva, P.; Nonell, A.; Gélabert, A.; Sonke, J. E.; Freydier, R.; Gainville, R.; Dupré, B.

519

Evidence of Zn isotopic fractionation in a soil-plant system of a pristine tropical watershed (Nsimi,

520

Cameroon). Chem. Geol. 2007, 239, 124–137.

521

17. von Blanckenburg, F.; von Wiren, N.; Guelke, M.; Weiss, D. J.; Bullen, T. D. Fractionation of

522

metal stable isotopes by higher plants. Elements 2009, 5 (6), 375–380.

523

18. Moynier, F.; Pichat, S.; Pons, M.-L.; Fike, D.; Balter, V.; Albarède, F. Isotopic fractionation

524

and transport mechanisms of Zn in plants. Chem. Geol. 2009, 267 (3–4), 125–130.

525

19. Arnold, T. I. M.; Kirk, G. J. D.; Wissuwa, M.; Frei, M.; Zhao, F. J.; Mason, T. F. D.; Weiss, D.

526

J. Evidence for the mechanisms of zinc uptake by rice using isotope fractionation. Plant Cell

527

Environ. 2010, 33 (3), 370–381.

528

20. Aucour, A. M.; Pichat, S.; Macnair, M. R.; Oger, P. Fractionation of stable zinc isotopes in the

529

zinc hyperaccumulator Arabidopsis halleri and nonaccumulator Arabidopsis petraea. Environ. Sci.

530

Technol. 2011, 45 (21), 9212–9217.

26


Page 26 of 34

Page 27 of 34


531

21. Caldelas, C.; Dong, S.; Araus, J. L.; Jakob Weiss, D. Zinc isotopic fractionation in Phragmites

532

australis in response to toxic levels of zinc. J. Exp. Bot. 2011, 62 (6), 2169–2178.

533

22. Jouvin, D.; Weiss, D. J.; Mason, T. F. M.; Bravin, M. N.; Louvat, P.; Zhao, F.; Ferec, F.;

534

Hinsinger, P.; Benedetti, M. F. Stable isotopes of Cu and Zn in higher plants: evidence for Cu

535

reduction at the root surface and two conceptual models for isotopic fractionation processes.

536

Environ. Sci. Technol. 2012, 46 (5), 2652–2660.

537

23. Tang, Y. T.; Cloquet, C.; Sterckeman, T.; Echevarria, G.; Carignan, J.; Qiu, R. L.; Morel, J. L.

538

Fractionation of stable zinc isotopes in the field-grown zinc hyperaccumulator Noccaea

539

caerulescens and the zinc-tolerant plant Silene vulgaris. Environ. Sci. Technol. 2012, 46 (18),

540

9972–9979.

541

24. Smolders, E.; Versieren, L.; Dong, S. F.; Mattielli, N.; Weiss, D.; Petrov, I.; Degryse, F.

542

Isotopic fractionation of Zn in tomato plants suggests the role of root exudates on Zn uptake. Plant

543

Soil 2013, 370 (1–2), 605–613.

544

25. Houben, D.; Sonnet, P.; Tricot, G.; Mattielli, N.; Couder, E.; Opfergelt, S. Impact of

545

root-induced mobilization of zinc on stable Zn isotope variation in the soil–plant system. Environ.

546

Sci. Technol. 2014, 48 (14), 7866–7873.

547

26. Deng, T. H. B.; Cloquet, C.; Tang, Y. T.; Sterckeman, T.; Echevarria, G.; Estrade, N.; Morel, J.

548

L.; Qiu, R. L. Nickel and zinc isotope fractionation in hyperaccumulating and nonaccumulating

549

plants. Environ. Sci. Technol. 2014, 48 (20), 11926–11933.

550

27. Couder, E.; Mattielli, N.; Drouet, T.; Smolders, E.; Delvaux, B.; Iserentant, A.; Meeus, C.;

551

Maerschalk, C.; Opfergelt, S.; Houben, D. Transpiration flow controls Zn transport in Brassica 27



552

napus and Lolium multiflorum under toxic levels as evidenced from isotopic fractionation. C. R.

553

Geoscience 2015, 347 (7–8), 386–396.

554

28. Arnold, T.; Markovic, T.; Kirk, G. J. D.; Schönbächler, M.; Rehkämper, M.; Zhao, F. J.; Weiss,

555

D. J. Iron and zinc isotope fractionation during uptake and translocation in rice (Oryza sativa)

556

grown in oxic and anoxic soils. C. R. Geoscience 2015, 347 (7–8), 397–404.

557

29. Wiederhold, J. G. Metal stable isotope signatures as tracers in environmental geochemistry.

558

Environ. Sci. Technol. 2015, 49, 2606–2624.

559

30. John, S. G.; Geis, R. W.; Saito, M. A.; Boyle, E. A. Zinc isotope fractionation during

560

high-affinity and low-affinity zinc transport by the marine diatom Thalassiosira oceanica. Limnol.

561

Oceanogr. 2007, 52 (6), 2710–2714.

562

31. Redjala, T.; Sterckeman, T.; Morel, J. L. Cadmium uptake by roots: contribution of apoplast

563

and of high- and low-affinity membrane transport systems. Environ. Exp. Bot. 2009, 67 (1), 235–

564

242.

565

32. Cloquet, C.; Carignan, J.; Libourel, G. Isotopic composition of Zn and Pb atmospheric

566

depositions in an urban/periurban area of northeastern France. Environ. Sci. Technol. 2006, 40 (21),

567

6594–6600.

568

33. Hacisalihoglu, G.; Hart, J. J.; Kochian, L. V. High- and low-affinity zinc transport systems and

569

their possible role in zinc efficiency in bread wheat. Plant Physiol. 2001, 125 (1), 456–463.

570

34. Fujii, T.; Moynier, F.; Blichert-Toft, J.; Albarède, F. Density functional theory estimation of

571

isotope fractionation of Fe, Ni, Cu, and Zn among species relevant to geochemical and biological

572

environments. Geochim. Cosmochim. Acta 2014, 140: 553–576. 28


Page 28 of 34

Page 29 of 34


573

35. Verbruggen, N.; Hermans, C.; Schat, H. Molecular mechanisms of metal hyperaccumulation in

574

plants. New Phytol. 2009, 181 (4), 759–776.

575

36. Degryse, F.; Shahbazi, A.; Verheyen, L.; Smolders, E. Diffusion limitations in root uptake of

576

cadmium and zinc, but not nickel, and resulting bias in the michaelis constant. Plant Physiol. 2012,

577

160 (2), 1097–1109.

578

37. Tang, D.; Morel, F. M. M. Distinguishing between cellular and Fe-oxide-associated trace

579

elements in phytoplankton. Mar. Chem. 2006, 98 (1), 18–30.

580

38. Redjala, T.; Sterckeman, T.; Skiker, S.; Echevarria, G. Contribution of apoplast and symplast to

581

short term nickel uptake by maize and Leptoplax emarginata roots. Environ. Exp. Bot. 2010, 68 (1),

582

99–106.

583

39. Aucour, A. M.; Bedell, J. P.; Queyron, M.; Magnin, V.; Testemale, D.; Sarret, G. Dynamics of

584

Zn in an urban wetland soil-plant system: coupling isotopic and EXAFS approaches. Geochim.

585

Cosmochim. Acta 2015, 160, 55–69

586

40. Sattelmacher, B. The apoplast and its significance for plant mineral nutrition. New Phytol.

587

2001, 149 (2), 167–192.

588

41. Fujii, T.; Albarède, F. Ab initio calculation of the Zn isotope effect in phosphates, citrates, and

589

malates and applications to plants and soil. PLoS ONE 2012, 7 (2), e30726. doi:

590

10.1371/journal.pone.0030726

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42. Zhao, F. J.; Shen, Z. G.; McGrath, S. P. Solubility of zinc and interactions between zinc and

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phosphorus in the hyperaccumulator Thlaspi caerulescens. Plant Cell Environ. 1998, 21 (1), 108–

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114. 29



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43. Jouvin, D.; Louvat, P.; Juillot, F.; Marechal, C. N.; Benedetti, M. F. Zinc isotopic fractionation:

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why organic matters. Environ. Sci. Technol. 2009, 43 (15), 5747–54.

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44. Estrade, N.; Cloquet, C.; Echevarria, G.; Sterckeman, T.; Deng, T.; Tang, Y.; Morel, J. L.

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Weathering and vegetation controls on nickel isotope fractionation in surface ultramafic

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environments (Albania). Earth Planet. Sci. Lett. 2015, 423, 24–35.

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Figure captions:

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Figure 1. Zn concentrations in root and shoot tissues (µg g-1, dry weight) of N. caerulescens (a) and

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T. arvense (b). F1: exchangeable root; F2: symplast root; F3: non-exchangeable root; For N.

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caerulescens, Zn-L-C: 1 µM Zn; Zn-H-C: 50 µM Zn; For T. arvense, Zn-L-A: 20 nM Zn; Zn-H-A:

606

5 µM Zn. (Data are the means ± SE of three replicates).

607 608

Figure 2. Zn isotope compositions in root fractions (a) and shoots (b) of N. caerulescens and T.

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arvense under different Zn treatments. For N. caerulescens, Zn-L-C: 1 µM Zn; Zn-H-C: 50 µM Zn;

610

For T. arvense, Zn-L-A: 20 nM Zn; Zn-H-A: 5 µM Zn. The root fractions include the exchangeable

611

root (F1, square), symplast root (F2, circle), nonexchangeable root (F3, triangle), and the whole root

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(diamond). (Data are the means ± SE of three replicates. Different letters on the same symbol

613

indicate that values were significantly different at P < 0.05 (Tukey’s HSD test))

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Figure 3. Zn isotope fractionation between the initial solution and whole plants (∆66ZnWhole plant–

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solution)

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Zn-L-C: 1 µM Zn; Zn-H-C: 50 µM Zn; For T. arvense, Zn-L-A: 20 nM Zn; Zn-H-A: 5 µM Zn.

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(Data are the means ± SE of three replicates. Different letters on the same symbol indicate that

619

values were significantly different at P < 0.05, Tukey’s HSD test). The isotopic composition of the

620

initial solution was –0.03‰.

and between root and shoot (∆66ZnShoot–

root)

in all Zn treatments. For N. caerulescens,

621

31



622 623

Figure 1

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624 625

Figure 2

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Figure 3

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Zinc Isotope Fractionation in the Hyperaccumulator ... - ACS Publications

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