Transformation and Immobilization of Chromium by Arbuscular

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Transformation and Immobilization of Chromium by Arbuscular Mycorrhizal Fungi as Revealed by SEM-EDS, TEM-EDS and XAFS Songlin Wu, Xin Zhang, Yuqing Sun, Zhaoxiang Wu, Tao Li, Yajun Hu, Dan Su, Jitao Lv, Gang Li, Zhensong Zhang, Lirong Zheng, Jing Zhang, and Baodong Chen Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b03659 • Publication Date (Web): 09 Nov 2015 Downloaded from http://pubs.acs.org on November 19, 2015

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Environmental Science & Technology

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Transformation and Immobilization of Chromium by Arbuscular Mycorrhizal Fungi as Revealed by SEM-EDS, TEM-EDS and XAFS

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Songlin Wu†,#,¶, Xin Zhang†,¶, Yuqing Sun†, #, Zhaoxiang Wu†, #, Tao Li†,

7

Yajun Hu†,⊥, Dan Su†,#, Jitao Lv‡, Gang Li‡, Zhensong Zhang‡, Lirong

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Zheng§, Jing Zhang§, Baodong Chen†,*

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†

State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental

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Sciences, Chinese Academy of Sciences, Beijing, 100085, People’s Republic of China

12

#

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University of Chinese Academy of Sciences, Beijing, 100049, People’s Republic of China

⊥

Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical

Agriculture, Chinese Academy of Sciences, Changsha, 410125, People’s Republic of China ‡

State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for

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Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, People’s Republic

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of China

18 19 20

§

Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of

Sciences, Beijing 100049, People’s Republic of China ¶

These authors contributed equally to this work.

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*Corresponding author, Baodong Chen, Phone: 0086-10-62849068; Fax: 0086-10-62923549;

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E-mail: [email protected]. 1

ACS Paragon Plus Environment


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TOC Art

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ABSTRACT:

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Arbuscular mycorrhizal fungi (AMF), as ubiquitous soil fungi that form symbiotic relationships

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with the majority of terrestrial plants, are known to play an important role in plant tolerance to

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chromium (Cr) contamination. However, the underlying mechanisms, especially the direct

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influences of AMF on translocation and transformation of Cr in the soil-plant continuum, are still

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unresolved. In a two-compartment root-organ cultivation system, the extraradical mycelium (ERM)

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of mycorrhizal roots was treated with 0.05 mmol L-1 Cr(VI) for 12 days to investigate the uptake,

32

translocation, and transformation of Cr(VI) by AMF using inductively coupled plasma mass

33

spectrometry (ICP-MS), scanning electron microscope equipped with energy dispersive

34

spectroscopy (SEM-EDS), transmission electron microscope equipped with energy dispersive

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spectroscopy (TEM-EDS) and X-ray absorption fine structure (XAFS) technologies. The results

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indicated that AMF can immobilize quantities of Cr via reduction of Cr(VI) to Cr(III), forming

37

Cr(III)-phosphate analogues, likely on the fungal surface. Besides, we also confirmed that the

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extraradical mycelium (ERM) can actively take up Cr [either in the form of Cr(VI) or Cr(III)], and

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transport Cr [potentially in the form of Cr(III)-histidine analogues] to mycorrhizal roots, but

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immobilize most of the Cr(III) in the fungal structures. Based on a XANES analysis of

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Cr(VI)-treated roots, we proposed that the intraradical fungal structures can also immobilize Cr

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within mycorrhizal roots. Our findings confirmed the immobilization of Cr by AMF, which plays

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an essential role in the Cr(VI) tolerance of AM symbioses.

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3



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INTRODUCTION

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Chromium (Cr) is a valuable metal commonly used in leather production, electroplating and steel

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manufacturing. Cr naturally exists in two stable forms, hexavalent chromium [Cr(VI)] and

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trivalent chromium [Cr(III)].1 Cr(VI) is highly mobile and more toxic to organisms than Cr(III).1, 2

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In recent decades, large amounts of Cr(VI) have been released into the environment by a wide

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range of industrial and agricultural activities, resulting in Cr contamination of soil and water.2 A

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recent survey of soil contamination in China showed that Cr is one of the eight key inorganic

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pollutants (Cd, Hg, As, Cu, Pb, Cr, Zn and Ni) in Chinese soils.3 Although Cr is an essential

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element for human beings and animals, excessive Cr can be toxic to all living organisms.4 As a

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non-essential element for plants, Cr usually disturbs plant physiological processes, including

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photosynthesis and respiration. Soil Cr contamination can produce negative effects on crop yield

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and threaten the food safety of human beings.5,6

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Plant rhizospheres usually serve as a favorable habitat for soil microorganisms, and some

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microorganisms can establish intimate relationships with plants. Arbuscular mycorrhizal fungi

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(AMF) are ubiquitous soil fungi that form symbiotic relationships with the majority of terrestrial

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plants.7 The fungi survive on carbohydrates from the host plants, and they in return provide

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mineral nutrients [especially phosphorus (P)] and water to their plant partner.7-9 Additionally, AMF

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can stabilize soil structure,10 relieve drought stress on plants,11 protect host plants from

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pathogens,12 and even take an active part in maintaining plant biodiversity and ecosystem

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stability.13 Many studies have also demonstrated that AM symbiosis plays an important role in

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plant resistance to contamination by heavy metals14, 15 such as As,16 Cd,17 Cu,18 Zn,19 Pb,20 Cr,21

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etc. Our recent work has shown that AM symbiosis can greatly enhance plant Cr tolerance, 4


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especially at high levels of Cr(VI) contamination.22

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However, to date, the underlying mechanisms of the enhanced plant Cr(VI) tolerance by AM

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symbioses are largely unknown. One potential mechanism is that AM symbiosis can improve plant

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growth via improving plant mineral nutrition (e.g., phosphorus, nitrogen, etc.), thereby indirectly

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enhancing plant Cr(VI) resistance. For example, in our recent study, AM symbiosis dramatically

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increased the dry weight of dandelion plants in Cr(VI)-contaminated soils by increasing plant P

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uptake.22 The larger plant biomass may dilute Cr in the plants, thus minimizing the Cr

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phytotoxicity, resulting in the so-called “growth dilution effect”.23 Another possible mechanism is

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that the extraradical mycelium (ERM) of mycorrhizal roots may directly reduce Cr(VI) to Cr(III),

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immobilize Cr(III) and restrict Cr(III) transfer to plants, similar to Cd24 and U.25,26 However, solid

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evidence is needed to test this hypothesis. Because Cr(VI) is much more toxic than Cr(III),

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organisms usually reduce Cr(VI) to Cr(III) to relieve Cr toxicity,27, 28, 29 and this reduction process

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may also occur in ERM. Besides, the ERM is known to have a high cation exchange capacity

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(CEC) and can retain large quantities of metals (especially those metal cations),30,31 thus

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obstructing the entrance of toxic metals into the root cytoplasm. Furthermore, even if the heavy

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metals taken up by the ERM are transported to the roots, the metals may not actually enter root

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cells across the symbiotic surface.24,32 Therefore, we hypothesized that the ERM can take up

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Cr(VI), and reduce it to Cr(III), then transport Cr(III) to mycorrhizal roots but retains most of the

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Cr(III) in its own structures. The next question is how AMF immobilize Cr(III). As Cr(III) can be

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complexed with phosphate, carboxylate, amine acids, thiols, hydroxide, etc., within organisms,33-35

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therefore, we further hypothesized that AMF diminish Cr(III) toxicity by combining Cr(III) with

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phosphate, thiols, histidine, or carboxylate. 5



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The objectives of this study were therefore to assess Cr(VI) uptake, translocation and

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transformation by AMF and to uncover the underlying mechanisms of enhanced plant resistance to

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Cr(VI) by AM symbiosis. A two-compartment root-organ cultivation system36 was adopted,

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through which we investigated not only the direct Cr(VI) absorption and translocation by ERM

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without interference from undesirable microorganisms but also obtained ERM material for further

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mechanism studies. Inductively coupled plasma mass spectrometry (ICP-MS) was used to

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determine Cr concentrations in the roots and the ERM, and synchrotron radiation micro-focused

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X-ray fluorescence (SR µ-XRF) was used to detect Cr in the ERM in situ. To localize Cr in the

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ERM and the mycorrhizal roots, a scanning electron microscope equipped with energy dispersive

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spectroscopy (SEM-EDS) and a transmission electron microscope equipped with energy

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dispersive spectroscopy (TEM-EDS) were used. Additionally, chemical speciation of Cr in the

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ERM was analyzed by using X-ray absorption fine structure (XAFS) spectroscopy with

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synchrotron radiation, which has developed in recent years into a powerful tool for the study of

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metal speciation in biological and environmental samples.29,33,37

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MATERIALS AND METHODS

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Root-Organ Cultures. Agrobacterium rhizogenes (Ri T-DNA)-transformed carrot (Daucus

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carotaL.) roots were cultivated in a minimal (M) medium38 solidified by 0.4% (w/v) phytagel

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(Sigma-Aldrich).36,39 The AM symbiosis in the solidified M medium was then established

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according to the methods described by St.-Arnaud et al.36 Briefly, the spores of the AM fungus

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Rhizophagus irregularis DAOM 197198 were first surface-sterilized with Tween 80 and

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chloramine T solutions, then rinsed in a solution of 1% (w/v) streptomycin sulfate and 0.5% (w/v) 6


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gentamycin sulfate, and finally spread on a 1.5% (w/v) water agar plate. The Ri

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T-DNA-transformed carrot roots were initiated on the modified M medium. Colonization was

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achieved by placing 10-15 germinated AMF spores near the apex of a 2-cm-long carrot root piece.

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The monoxenic culture was then incubated in the dark at 25℃. After 3 months, approximately

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4,000 spores associated with approximately 80 mg (dry weight) of roots had developed in each

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Petri plate. The transformed carrot roots without AMF inoculation were also maintained as

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experimental controls.

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Before the formal experiment, the Cr concentrations in solidified and liquid M media and

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nonmycorrhizal and mycorrhizal roots were determined using an ICP-MS (7500a Agilent

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Technologies, California, USA) after digestion by HNO3. The Cr concentrations were as follows:

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solidified M medium, 1.92×10-4 mmol L-1; liquid M medium, 1.92×10-4 mmol L-1; nonmycorrhizal

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roots, 0.57 mg kg-1 (dry weight); mycorrhizal roots, 0.39 mg kg-1 (dry weight).

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Experimental Design. Two-compartment Petri plates (9 cm in diameter, Figure S1)36 were used

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for the formal experiment. Cultures were initiated in a root compartment composed of 30 mL M

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medium gelled with 0.4% (w/v) phytagel. The counterpart hyphal compartment allowed only

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growth of extraradical hyphae, allowing us to investigate the direct interactions of hyphae with

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Cr(VI).

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We tested two root compartment treatments: mycorrhizal or nonmycorrhizal. For the mycorrhizal

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treatment (“+M”), a 2-cm2 piece of solid M medium containing carrot roots colonized by

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Rhizophagus irregularis was introduced, whereas for the nonmycorrhizal treatment (“-M”), a

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2-cm2 piece of solid M medium containing uninoculated carrot roots was introduced.39 The initial

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dry weight of both mycorrhizal and nonmycorrhizal roots was approximately 0.90 mg. The Petri 7



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plates were incubated horizontally in an inverted position at 25℃ in the dark for 6 weeks. The

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Petri plates were then set upright for 3 additional weeks, and the hyphal compartments were filled

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with 10 mL liquid M medium without sucrose and phytagel. The extraradical mycelium (ERM) of

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the “+M” treatment started to cross the central wall between the root compartment and the hyphal

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compartment and proliferated in the liquid medium. The cultures were examined weekly, and the

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roots that crossed the central wall were trimmed to prevent their growth into the hyphal

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compartment. After three additional weeks, the cultures were ready for the formal experiment.

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Cr(VI) Treatment. For each“-M” and “+M” treatment, we applied two treatments in the hyphal

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compartment: additions with or without Cr(VI) (i.e., the “+Cr” or “-Cr” treatments). In the “+Cr”

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treatment, 10 mL of liquid M medium without sucrose, vitamins, potassium iodide, phosphate and

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EDTA-Fe (in the form of NaFeEDTA) [to avoid potential Cr(VI) reduction by vitamins or

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potassium iodide, and Cr(III) precipitation by phosphate, or complexation with EDTA in the

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medium] but with Cr(VI) (in the form of K2CrO4 at a concentration of 0.05 mmol L-1; the pH was

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adjusted to 5.5) was added to the hyphal compartment after removal of the old medium via pipette.

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Similarly, 10 mL of the same liquid M medium without Cr(VI) but with the same amount of K (in

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the form of KCl) as the K2CrO4 in the “+Cr” treatment was applied in the “-Cr” treatment. To

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investigate whether Cr(VI) uptake and translocation by hyphae was an active process and whether

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Cr(VI) transformation by AMF was a metabolic process, two control treatments were arranged.

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For one control, metabolic activity of the hyphae was inhibited by formaldehyde (2% v/v) for 24 h

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according to Rufyikiri et al.25 After 24 h, we removed the formaldehyde solution and washed the

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hyphae carefully with Milli-Q water before Cr(VI) was applied to avoid direct interaction between

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formaldehyde and Cr(VI). In the second control, the hyphal activity was inhibited by 0.5 mmol L-1 8


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2,4-dinitrophenol (DNP), a respiration inhibitor and an uncoupler of oxidative phosphorylation,

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which can cause dissipation of the proton motive force across membranes, thereby inhibiting

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active metal uptake by fungi.40 By comparing the Cr concentrations in the DNP- and

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Cr(VI)-treated hyphae with the Cr concentrations in the solely Cr(VI)-treated hyphae, we aimed to

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identify whether Cr became adsorbed onto the fungal surface. Therefore, we performed a total of 6

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treatments, as shown in Table 1. Treatments “-M-Cr” [noninoculation in the root compartment and

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no Cr(VI) addition in the hyphal compartment] and “-M+Cr” [noninoculation in the root

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compartment and 0.05 mmol L-1 Cr(VI) addition in the hyphal compartment] were replicated 8

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times, whereas treatments “+M-Cr” [inoculation in the root compartment and no Cr(VI) addition

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in the hyphal compartment], “+M+Cr” [inoculation in the root compartment and 0.05 mmol L-1

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Cr(VI) addition in the hyphal compartment], “+M+CrF” [inoculation in the root compartment and

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0.05 mmol L-1 Cr(VI) addition in the hyphal compartment after addition of ERM inhibition by 2%

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(v/v) formaldehyde] and “+M+CrD” [inoculation in the root compartment and 0.05 mmol L-1

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Cr(VI) plus 0.5 mmol L-1 DNP addition in the hyphal compartment] were replicated twelve times

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(with four treatments primarily for microscopy observations and spectroscopy studies). The Petri

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plates with the various treatments were arranged in a completely randomized order and incubated

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in the dark at 25℃ for 12 days.

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Assessment of Variables. By the end of the experiment, the total number of spores in the hyphal

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compartment and the root compartment were assessed using 1-cm grids marked on the bottom of

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each Petri plate.25 The roots in the root compartment were separated from the medium by

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solubilizing the solidified M media in 10 volumes of citrate buffer (pH 6.0), and the roots were

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then washed carefully with Milli-Q water. The ERM and liquid M medium in the hyphal 9



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compartment were collected simultaneously. The ERM samples were washed thoroughly, first

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with cold 0.5 mmol L-1 Ca(NO3)2 (4℃) for 10 min to remove apoplastic Cr and then with Milli-Q

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water. A small portion of the fresh roots and hyphae were used for microscopy observations and

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determination of mycorrhizal colonization, and the remaining portions were frozen in liquid

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nitrogen and stored at -80℃ for subsequent ICP-MS and spectroscopy analysis.

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The collected liquid M medium from the hyphal compartment was filtered through a 0.45-µm

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millipore filter. The pH of the medium was determined by a pH meter (FE20-FiveEasy Plus,

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Mettler Toledo, Zurich, Switzerland). Cr(VI) was analyzed by the Cr(VI)-specific colorimetric

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reagent 1,5-diphenylcarbazide (DPC) method.41 The total Cr concentrations were determined with

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an inductively coupled plasma atomic emission spectrometer (ICP-AES, Prodigy, Leemans, New

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hampshire, USA) after acidification by HNO3. The Cr(III) concentration was calculated as the

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difference between the total Cr concentration and the Cr(VI) concentration.

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The root and ERM samples that were stored at -80℃ were lyophilized with a freeze dryer at -50℃

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for 48 h. The dried samples were motor-homogenized in liquid nitrogen after weighing. To

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analyze the Cr concentrations, dried root and hyphal samples were digested in HNO3 using a

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microwave-accelerated reaction system (Mars 5, CEM Microwave Technology Ltd., Matthews,

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North Carolina, USA) in a three-step digestion program. The temperature was raised to 120℃ over

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8 min, held for 3 min, then raised to 160℃ over 11 min, held for 7 min, and finally raised to 180℃

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over 8 min and held for 15 min. The digested solutions were then held at 140℃ for 4 h to remove

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the nitric acid. The dissolved samples were then diluted to 10 mL with Milli-Q water. The Cr

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concentrations were determined using an ICP-MS (model 7500a, Agilent Technologies, California,

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USA), and the P concentrations were determined using an ICP-AES (Prodigy, Leemans, New 10


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hampshire, USA). Blanks and internal standards of bush leaves (GBW07603, China Standard

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Research Center) and tea leaves (GBW10016, China Standard Research Center) were used to

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ensure the accuracy of the chemical analyses.

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To determine AMF colonization, samples of fresh roots were cleared in 10% KOH and stained

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with Trypan blue following a modified procedure of Phillips and Hayman42 and omitting phenol

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from the solutions. The intensity of the mycorrhizal colonization on the root system (M%) was

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determined by the method of Trouvelot et al.(1986) 43 using “mycocalc”software.

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Scanning Electron Microscopy (SEM) Analysis. Morphological changes in the hyphae exposed

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to Cr(VI) were examined using SEM. Fresh hyphae in the hyphal compartment of the treatments

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“+M+Cr”

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piperazine-1,4-bisethanesulfonic acid (PIPES) (Amresco 0169, Ohio, USA) buffer solution (pH

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7.2) overnight and then thoroughly washed 3 times with the same buffer solution. The hyphae

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were then treated with 1% osmium tetroxide for 2 h and dehydrated in a graded acetone series

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(30%, 50%, 70%, 80%, 90%, 100%). Then, the mycelia were dried, sputter-coated and analyzed

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using a field emission scanning electron microscope equipped with an energy dispersive X-ray

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spectrometer (FE-SEM-EDS, SU-8020, Hitachi, Tokyo, Japan).

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Synchrotron Radiation Micro-focused X-Ray Fluorescence (SR µ-XRF) Analysis. To detect

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Cr in the hyphae in situ without digestion by HNO3, SR µ-XRF analyses were performed. Fresh

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hyphae in the hyphal compartment of the treatments “+M+Cr” and “+M-Cr” were attached to 3M

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tape (cat. 810, Minnesota Mining and Manufacturing Company, Minn., USA) and freeze dried at

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-25℃ for 72 h. The SR µ-XRF analyses were performed at beamline 4W1B, Beijing Synchrotron

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Radiation Facility (BSRF). The detailed procedure is described in the Supporting Information (SI).

and

“+M-Cr”

were

fixed

with

2.5%

11


glutaraldehyde

in

a


221

Transmission Electron Microscope (TEM) Analysis. The TEM analyses were performed to

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preliminarily identify the locations of Cr at the subcellular level in the mycorrhizal roots in

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treatment “+M+Cr”, in which only the living ERM in the hyphal compartment was treated with

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Cr(VI). Fresh roots in the root compartment of treatment “+M+Cr” were fixed overnight with 2.5%

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glutaraldehyde in a boric acid buffer solution (pH 7.4) and then thoroughly washed 3 times with

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the same buffer solution, followed by fixation with 1% osmium tetroxide for 2 h. Then, the roots

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were washed 3 times with the boric acid buffer, dehydrated in a graded acetone series (30%, 50%,

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70%, 80%, 90%, 100%), and finally embedded in Spurr's resin. Ultrathin sections (90 nm) were

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obtained using a Leica Ultracut UCT (Leica, Solms, Germany) with a glass knife. The sections

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were positioned on copper grids, stained with uranyl acetate and lead citrate, and observed under a

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transmission electron microscope (TEM, H-7500, Hitachi, Tokyo, Japan) operating at 80 keV. The

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TEM-EDS spectra were collected on a TEM (JEM-2011, JEOL, Tokyo, Japan) equipped with an

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energy dispersive X-ray spectrophotometer. More than four sections cut from different roots were

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examined.

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X-Ray Absorption Fine Structure (XAFS) Spectroscopy Analysis. The lyophilized and

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homogenized samples were used for XAFS analysis to detect Cr speciation. Roots from the root

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compartment of treatment “+M+Cr” were pressed into thin slices with a diameter of 10 mm and a

238

thickness of 2 mm and then attached to 3M tape. The ERM samples from the treatments “+M+Cr”,

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“+M+CrF” and “+M+CrD” were attached directly and uniformly to 3M tape. The XAFS spectra

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of the ERM and root samples were collected on beamline 1W1B at the Beijing Synchrotron

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Radiation Facility (BSRF). Detailed methods are described in the SI.

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Cr Speciation in Mycorrhizal Roots Exposed to Cr(VI): The XANES Study. The XANES 12


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experiment was a supplement to the above XAFS study of the ERM exposed to Cr(VI) and aimed

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to investigate Cr speciation changes in the roots after AMF colonization. Petri plates 9 cm in

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diameter were filled with 40 mL solidified M medium with an addition of 0.02 mmol L-1 Cr(VI).

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Mycorrhizal and nonmycorrhizal roots were then introduced separately, resulting in 2 treatments

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with 8 replicates each. All cultures were incubated in the dark at 25°C for 3 months. The roots

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were then collected and lyophilized. The Cr concentrations were determined via ICP-MS, and the

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Cr speciation was analyzed via XANES.

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Statistical Analysis. Data on the AM development parameters and Cr concentrations in biological

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samples were subjected to one-way analysis of variance (ANOVA) tests, followed by Duncan’s

252

test (p < 0.05), to determine the significance of the differences between the treatments.

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RESULTS AND DISCUSSION

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AM Development. The intensity of the mycorrhizal colonization on the root systems (M%) was

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generally greater than 40%, and no differences were observed among the inoculated treatments

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(Table S1). The root dry weights ranged from 20 to 45 mg in the root compartment, and AM

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symbiosis correlated with higher root biomass (p < 0.05) but not with higher plant phosphorus (P)

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concentration (Table S2). The greater root growth may due to the beneficial effects of AM

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symbiosis because AM symbiosis can enhance plant uptake of nitrogen and certain micronutrients,

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such as Cu, Zn, etc.7 The spore numbers in the root compartment of inoculated treatments were

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between 2000 and 2500, and no differences were observed among the treatments. Numerous

263

hyphae crossed the barrier between the two compartments and developed in the hyphal

264

compartment. The mycelium dry weight in the hyphal compartment was greater than 1.90 mg. The 13



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spore numbers in the hyphal compartment were greater than 1000 per plate with no significant

266

differences among the inoculated treatments with the exception of the decreased spore numbers

267

due to Cr(VI) stress (Table S1, p < 0.05). In the noninoculated control treatments, no mycorrhizal

268

colonization was observed; thus, no ERM or spores were present in the hyphal compartment

269

(Table S1).

270

Starting at an initial pH of 5.5, the pH in the hyphal compartment increased significantly in the

271

treatments “+M-Cr” and “+M+Cr” (Table S1, p < 0.05). However, the pH in the hyphal

272

compartment did not change when the ERM was treated with formaldehyde or DNP. The dramatic

273

alkalization of the medium associated with ERM development may result from the active uptake

274

of NO3--N, including the NO3-/H+ symport or NO3–/OH– antiport mechanisms exerted by the

275

fungus. These processes likely overwhelm any other ERM-promoted acidification, leading to a net

276

alkalinization.44,45 The formaldehyde and DNP inhibited this process by inhibiting hyphal activity;

277

thus, the pH of the medium did not change. These results also confirm the effectiveness of the two

278

inhibitors on AMF activity.

279

Cr Concentrations in the Hyphal Compartment. In the treatments “-M-Cr” and “+M-Cr”, Cr

280

was not detected via ICP-AES in the hyphal compartment, demonstrating that no Cr

281

contamination was present in the cultivation system (Figure 1). Compared with the control

282

treatment “-M+Cr”, the total Cr concentrations in the hyphal compartments of the treatments

283

“+M+Cr”, “+M+CrF” and “+M+CrD” were significantly lower (Figure 1, p< 0.05), indicating

284

possible uptake/sorption of Cr by the ERM. Moreover, in the hyphal compartment of the

285

inoculated treatments, some Cr(VI) was reduced to Cr(III) (Figure 1), especially for treatment

286

“+M+CrF”, in which nearly half of the residual Cr(VI) was reduced to Cr(III). This reduction 14


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likely resulted from the high reduction abilities of dissolved organic carbons (e.g. polysaccharides,

288

peptides and glycoproteins etc) derived from the fungal biomass (especially the dead or

289

inactivated fungal biomass).46 Alternatively, the Cr(VI) may have been reduced by extracellular

290

soluble reductase excreted by the fungi, similar to that of certain Gram-negative bacteria.47

291

Another possibility is that Cr(VI) was firstly reduced in the fungi or on the fungal surface by

292

chromate reductase or intracellular compounds (e.g., cysteine, glutathione and sulfite), then some

293

Cr(III) in the fungal biomass was released into the growth medium due to hyphal break or

294

complexation of Cr(III) with complexing ligands.48

295

The ERM Adsorbs, Accumulates, and Actively Transports Cr to Mycorrhizal Roots. Figure

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2A shows that the ERM treated with Cr(VI) can accumulate more than 1000 mg kg-1 Cr. The SR

297

µ-XRF analysis also showed high Cr signals in Cr(VI)-treated in situ ERM (Figure S2),

298

corroborating the results from the ICP-MS. These results are similar to the results from a previous

299

study that documented large accumulations of Cu and Zn in AMF biomass.31 Moreover, the total

300

Cr concentrations in the hyphal compartment solution of the treatments “+M+CrF” and “+M+CrD”

301

were significantly lower than the total Cr concentrations in “+M+Cr” (Figure 1, p < 0.05).

302

Accordingly, the inactivated ERM in the hyphal compartment contained higher Cr concentrations

303

than the living hyphae treated with the same quantity of Cr(VI) (Figure 2A, p < 0.05). Just as

304

mentioned above, Cr(VI) was probably reduced to Cr(III) in the medium, in the fungi or on the

305

fungal surface by chromate reductase or compounds that can serve as electron donors (e.g.,

306

dissolved organic carbons, cysteine, glutathione or sulfite).48 The Cr concentrations in the

307

inactivated hyphae were likely associated with Cr adsorption by the fungal biomass, as the

308

inactivated hyphae exhibited a higher adsorption capacity than living hyphae.30 Similar to 15



309

previous studies on Serratia spp.49 and Rhizopus arrhizus,50 extracellular polymeric substances

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(EPSs), including carboxyl or phosphate groups, may play an important role in Cr(III) adsorption.

311

On the other hand, it is essential to point out that Cr(VI) may first be bound to the positively

312

charged complexing groups on fungal surface, and then reduced to Cr(III) by adjacent

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electron-donor compounds (such as glutathione, cysteine, amino acid, etc)51. This occurs

314

especially under acid conditions, as low pH can make the biomass surface more positive.

315

Therefore the Cr(VI) adsorption may preferably occur on the surface of inactivated AM fungi, as

316

they maintained an acid condition (the pH was 5.3-5.4) (Table S1). The SEM observation showed

317

that the mycelium without Cr(VI) stress was rod shaped and had a smooth surface (Figure 3A).

318

However, when treated with Cr(VI), the hyphae became rougher with small irregular structures on

319

the surface (Figure 3B), which may result from EPS production. The EDS spectrum also

320

supported the presence of Cr on the edge (surface) of mycelia treated with Cr(VI) (Figure 3c),

321

whereas no Cr signal was detected in the mycelia free of Cr(VI) treatment (Figure 3a and Figure

322

3b).

323

The root Cr concentrations were higher in treatment “+M+Cr” than in other treatments, including

324

“+M+CrF” and “+M+CrD” (Figure 2B, p < 0.05), which confirms that living ERM likely can take

325

up and transfer Cr to mycorrhizal roots via a process at least partially metabolic.

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To reveal the pattern of Cr partitioning between the ERM and the roots, we calculated the

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percentage of Cr content in the ERM in the hyphal compartment relative to the total Cr content in

328

the ERM and mycorrhizal roots (Table S3). The majority of the Cr was immobilized in the fungal

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biomass: more than 70% of the Cr taken up or adsorbed by the ERM was retained in the ERM,

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and less than 30% was translocated to the mycorrhizal roots. 16


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TEM-EDS Study of Cr in Mycorrhizal Roots. Using ICP-MS analyses of the Cr concentrations

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in roots from different treatments (Figure 2B), we found that the ERM can actively transport Cr

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from the hyphal compartment to the mycorrhizal roots in the root compartment. However, it was

334

unclear whether Cr was actually transferred to the root cells. Like Cd, which was found to be

335

retained in the fungal structures of mycorrhizal Lotus japonicus,24 we hypothesized that the Cr

336

taken up by the ERM was retained primarily in the fungal structures of the mycorrhizal roots. The

337

TEM image showed that certain root cells in the mycorrhizal roots in the root compartment

338

contained arbuscules or intracellular mycelium (Figures 4D and 4E). The EDS data indicated a

339

high Cu signal in the root samples, which may be due to the copper grid used to hold the samples.

340

Although the Cr concentration was low in the mycorrhizal roots, Cr Kα peaks (5.41 keV) were

341

found in both the fungal structures and the nearby root cell cytoplasm (Figures 4a and 4b) but not

342

in the background area. Moreover, the fungal structures tended to have a higher Cr peak (5.41 keV)

343

(with 162 net counts) than the cytoplasm of plant cells (with 110 net counts), potentially indicating

344

Cr immobilization by the fungal structures within the mycorrhizal roots. However, EDS has its

345

own drawbacks with respect to metal mapping, i.e., its low resolution and high detection limit.

346

Therefore, the subcellular localization of Cr via more advanced techniques with higher resolutions

347

and lower detection limits is urgently needed. Regardless, the present TEM-EDS analysis

348

confirms the presence of Cr in the mycorrhizal roots, thus corroborating the ICP-MS results

349

showing that the ERM can actively transport Cr from a distance to the mycorrhizal roots.

350

Reduction of Cr(VI) to Cr(III)-Phosphate Analogues by the Hyphae. Normalized Cr-K-edge

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X-ray adsorption near-edge spectroscopy (XANES) spectra of standard chemical compounds and

352

the hyphal samples from different treatments are shown in Figure S3. The Cr(VI) standard 17



353

compounds (K2CrO4 and K2Cr2O7) exhibited a peak at 5993.3 eV, which is significantly different

354

from the Cr(III) standard compounds that produced no peak at 5993.3 eV. The pre-edge feature of

355

Cr(VI) was attributed to the transition of 1s electrons to an empty p–d hybridized orbit of Cr(VI)

356

in a tetrahedral coordination.52 Based on the measurement of this peak, the Cr(III)/Cr(VI) ratio can

357

be assessed in the mixture.53 No hyphal samples exhibited a peak at 5993.3 eV, indicating that Cr

358

in the ERM or on fungal surface was in the form of Cr(III). Same to our previous assumption,

359

Cr(VI) may be reduced by certain enzymes54 or specific biochemical groups (electron donors, e.g.

360

glutathione, carboxyl and amino groups).51 The high Cr(VI) reduction capability of inactivated

361

hyphae demonstrates that Cr(VI) reduction can be a nonmetabolic process,55 possibly resulting

362

from dissolved organic carbons derived from the AMF.46 Because DNP inhibited active Cr(VI)

363

uptake by inhibiting the respiration process,40 the Cr(III) adsorbed on the fungal surface indicated

364

that Cr(VI) reduction likely occur on the fungal surface, or in the hyphosphere and adsorbed on

365

fungal surface.51, 56, 57 Besides, considering the fact that a small proportion of Cr (about 26.4%)

366

was transported to mycorrhizal roots via living ERM (Figure S3) and the root Cr concentration

367

decreased when the ERM in hyphal compartment was inactivated by formaldehyde or DNP

368

(Figure 2), we predict that the living hyphae may also absorb a small proportion of Cr either in the

369

form of Cr(VI) [through sulfate transporter,58 once Cr(VI) is taken up, it will be reduced to Cr(III)

370

immediately in the fungi by enzymes or biochemical groups] or Cr(III) (through iron uptake

371

system59), and Cr(III) is then transported to mycorrhizal roots.

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To obtain quantitative information regarding Cr speciation in the fungal hyphae, a principal

373

component analysis (PCA) and a linear combination ﬁtting (LCF) analysis were performed on the

374

normalized XANES spectra. The PCA provided a statistical basis for selecting the proper Cr 18


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species for inclusion in the LCF model.60 The results suggested that Cr(III)-phosphate,

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Cr(III)-nitrate, Cr(III)-sulfate, Cr(III)-cysteine, Cr(III)-histidine and Cr(III)-acetate could be

377

selected for LCF analysis because these compounds showed low Chi Sq(

Transformation and Immobilization of Chromium by Arbuscular

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