Coupling between nitrogen fixation and tetrachlorobiphenyl

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Coupling between nitrogen fixation and tetrachlorobiphenyl dechlorination in a rhizobium-legume symbiosis Xiaomi Wang, Ying Teng, Chen Tu, Yongming Luo, Chris Greening, Ning Zhang, Shixiang Dai, Wenjie Ren, L ZHAO, and Zhengao Li Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05667 • Publication Date (Web): 24 Jan 2018 Downloaded from http://pubs.acs.org on January 25, 2018

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Environmental Science & Technology

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Coupling between nitrogen fixation and tetrachlorobiphenyl dechlorination in a

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rhizobium-legume symbiosis

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Xiaomi Wang1,4, Ying Teng1*, Chen Tu2, Yongming Luo1,2*, Chris Greening3, Ning

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Zhang1, Shixiang Dai1,4 , Wenjie Ren1, Ling Zhao1, Zhengao Li1

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1 Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil

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Science, Chinese Academy of Sciences, Nanjing 210008, P.R. China

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2 Key Laboratory of Coastal Environmental Processes and Ecological Remediation,

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Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai

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264003, P.R. China

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3 School of Biological Sciences, Monash University, Clayton, Victoria 3800,

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Australia

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4 University of Chinese Academy of Sciences, Beijing 100049, P.R. China

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*Corresponding author:

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E-mail: [email protected] (Y. Teng); [email protected] (Y. M. Luo)

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Phone: +86-25-86881531; +86-535-2109007；

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Fax: +86-25-86881126; +86-535-2109000.

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ACS Paragon Plus Environment


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ABSTRACT

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Legume-rhizobium symbioses have the potential to remediate soils contaminated with

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chlorinated organic compounds. Here, the model symbiosis between Medicago sativa

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and Sinorhizobium meliloti was used to explore the relationships between symbiotic

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nitrogen fixation and transformation of tetrachlorobiphenyl PCB 77 within this

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association. 45-day-old seedlings in vermiculite were pre-treated with 5 mg L-1 PCB

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77 for 5 days. In PCB-supplemented nodules, addition of the nitrogenase enhancer

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molybdate significantly stimulated dechlorination by 7.2-fold, and reduced tissue

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accumulation of PCB 77 (roots by 96% and nodules by 93%). Conversely,

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dechlorination decreased in plants exposed to a nitrogenase inhibitor (nitrate) or

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harboring nitrogenase-deficient symbionts (nifA mutant) by 29% and 72%,

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respectively. A range of dechlorinated products (biphenyl, methylbiphenyls,

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hydroxylbiphenyls, and trichlorobiphenyl derivatives) were detected within nodules

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and roots under nitrogen-fixing conditions. Levels of nitrogenase-derived hydrogen

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and leghemoglobin expression correlated positively with nodular dechlorination rates,

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suggesting a more reducing environment promotes PCB dechlorination. Our findings

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demonstrate for the first time that symbiotic nitrogen fixation acts as a driving force

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for tetrachlorobiphenyl dechlorination. In turn, this opens new possibilities for using

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rhizobia to enhance phytoremediation of halogenated organic compounds.

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Table of Contents (TOC)

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1. Introduction

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Rhizobia-legume symbioses are widely distributed and account for a quarter of total

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natural terrestrial N2 fixation annually. 1-3 In these symbioses, rhizobial colonization

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of plant root cells induces root nodule development and bacterial differentiation into

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bacteroids. 3 Rhizobia use the molybdenum (Mo)-dependent nitrogenase to fix N2

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within nodules, while plant nodule cells synthesize leghemoglobin (LegHb) to

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maintain a microoxic environment that protects this enzyme from oxygen activation. 3

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In addition to their role in N2 fixation, rhizobia-legume associations have recently

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attracted interest for their capacity to remediate sites contaminated with organic

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pollutants. Most notable is their capacity to biodegrade polychlorinated biphenyls

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(PCBs), 4 a group of persistent organic pollutants classified as human carcinogens and

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environmental toxins. 5 Although the production and application of these compounds

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was banned by the 1980s, their persistence and bioaccumulation continues to pose

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health and environmental risks. 6, 7 It has been through both laboratory experiments

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and field studies that rhizobia-alfalfa symbioses are highly effective in remediating

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PCB contamination in soils. Thus, harnessing this symbiosis may prove a

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cost-effective and environmentally attractive way to remove contaminants. 8-10

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There is growing evidence that collaboration between plant and symbiotic bacteria is

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crucial for effective transformation of xenobiotics. 11 Specifically, legumes sequester

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and accumulate PCBs from soils (phytoextraction) and their rhizobial endophytes in

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turn mediate their dechlorination and degradation (rhizoremediation). 4 The legume 4 / 27


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alfalfa (Medicago sativa) is a particularly promising candidate for phytoextraction due

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to its extensive root systems, fast growth rate, and environmental adaptability. 4, 5

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Reflecting its high tolerance, the species can accumulate PCBs without exhibiting

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severe stress symptoms. 12, 13 In contrast to several plant species such as poplar and

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maize, 14, 15 alfalfa cells are unable to transform most organochlorine compounds. 16

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However, rhizobia colonizing their root nodules rapidly dechlorinate certain PCBs, in

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turn reducing their dioxin-like toxicity and rendering them more readily degradable

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through oxidative processes. 4, 9, 17 It is proposed that the elaborate cooperation and

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communication between host and microbe results in synergistic PCB remediation, for

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example by enhancing pollutant bioavailability for the rhizosphere and alleviating

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oxidative stress on plant cells. 4, 5, 8-10, 18 Importantly, utilization of symbiotic partners

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equipped with appropriate biodegradative capabilities is necessary to optimize

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phytoremediation. Reflecting this, Mehmannavaza et al. (2002) reported that

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augmentation with non-degradative Sinorhizobium strain contributed little to PCB

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depletion in alfalfa, especially after plants were fully developed. 18 However, several

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rhizobial strains have been reported that can degrade PCBs both in culture and within

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root nodules. 9, 19, 20 Most notably, we have shown that inoculation with Sinorhizobium

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meliloti strain NM significantly promotes alfalfa performance and PCB removal from

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soils in both laboratory and field settings. 8, 10

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To data, limited attention has been paid to rhizobial PCB degradation within plant

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tissues (especially within nodules) and the dynamic interactions this process may have 5 / 27



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with plant metabolism. Considering the complex metabolism and intimate cooperation

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of host and microbe during symbiosis, it is possible that microbial biotransformation

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of PCBs and other pollutants might be influenced by plant metabolism and signaling.

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4, 17

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pollutant degradation would deepen our understanding of the underlying mechanisms

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involved in rhizobial remediation. As a precedent, it has been demonstrated that, in

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some halorespiring anaerobes, N2 fixation activity gives rise to higher rates of

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reductive dechlorination against chloroethylene, though the mechanistic basis for this

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is unclear. 21

In particular, knowledge of the relationship between symbiotic N2 fixation and

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In this study, we used the model PCB-degrading strain S. meliloti NM to investigate

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the remediation of 3,3’,4,4’-tetrachlorobiphenyl (PCB 77) within N2-fixing root

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nodules.8, 9, 17 We hypothesized that nodules serve as a “micro-reactor” to support the

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accumulation and transformation of PCB 77. We investigated whether the metabolism

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of PCB 77 was influenced by N2 fixation and determined what degradation products

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were produced. N2 fixation activity within nodules was manipulated by two

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approaches: i) treatment with nitrogenase inhibitors (NO3− or NO2−) or an enhancer

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(MoO42−), and ii) inoculation with a bacterial nifA mutant to render nitrogenase

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inactive.

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2. Materials and Methods

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2.1. Chemicals 6 / 27


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Standards of PCB 77, biphenyl, 4-methyl-biphenyl, 2,2’-dimethyl-biphenyl,

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4-(1-methylethyl)-biphenyl (99% purity), and 4-methoxy-2,2',5'-trichlorobiphenyl

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were purchased from Accustandard (New Haven, CT). n-Hexane

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(chromatography-grade purity) used for GC analysis was procured from Tedia

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Company (Fairfield, OH). The remaining chemicals and reagents were purchased

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from Sigma-Aldrich (St Louis, MO) and were of analytical reagent grade or higher

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purity unless stated otherwise.

115 116

2.2. Microorganisms

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The other strains and plasmids used are listed in Table S1. S. meliloti strain NM

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(wild-type strain, WT), obtained from the Agricultural Culture Collection of China

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(ACCC17519), was grown in yeast mannitol (YM) agar plates at 28 °C. 9, 17 For

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rhizobial inoculation, cells were grown to exponential phase in YM broth medium at

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28 °C on a rotary shaker at 180 rpm min−1. The bacteria were then harvested by

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centrifugation, washed, and resuspended in sterile water (2 × 109 cell mL−1) for

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inoculation. 17 To demonstrate rhizobial colonization, the plasmid pHC60 carrying a

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green fluorescent protein (GFP) was mobilized into S. meliloti NM via a triparental

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mating procedure (Supplementary Text).

126 127

To render nitrogenase inactive, a nifA mutant (strain Smy) was constructed by the

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insertion of a gentamycin resistance plasmid into nifA (Supplementary Text). nifA

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activates expression of the nifHDK operon, which encodes the structural genes of 7 / 27



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Mo-nitrogenase. 3 A NarA mutant that compromised the expression of nitrate

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reductase catalytic subunit (NapA) was constructed following the same procedure.

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2.3. Inoculation experiments

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Inoculations were performed to investigate the dynamic interplay between rhizobial

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N2 fixation and biotransformation of PCB 77 in nodules of alfalfa. Alfalfa (Medicago

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sativa) seeds were surface sterilized and germinated at 25 °C in the dark. For infection,

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seeds were dipped into a suspension of S. meliloti (strain WT or Smy), and

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subsequently germinated. 10 For the non-inoculated group (alfalfa), sterilized

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deionized water was applied. Uniform seedlings were transferred to modified Leonard

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jars (glass, double layer; Fig. S1), which were fully wrapped with aluminum foil and

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Parafilm to prevent photolysis and volatilization of any added PCB 77. The internal

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layer was filled with sterile vermiculite and the external layer contained 150 mL of

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nitrogen-free Fahraeus solution. Fifteen plants were grown in the same chamber.

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Plants were irrigated three times a week and grown in an illuminating incubator under

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a photoperiod of 16-h light/8-h dark (25/18°C day/night) and a photon flux density of

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80 µmol m−2 s−1.

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At 45 days post rhizobial inoculation, 150 µL of PCB 77 stock solution (final

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concentration 5 mg L-1) was added where specified and incubated for 5 days (to reach

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equilibrium in plant roots). 13 The relatively high concentration of 5 mg L-1 PCB 77

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was selected instead of typical environmental concentrations, given dechlorination 8 / 27


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and transformation phenomena would be more readily detectable. In order to avoid

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differential dispersion of PCB 77 in the solid phase (vermiculite), the stock was added

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to the external layer of Fahraeus solution. To evaluate the interaction between

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nitrogenase activity and rhizobial transformation of PCB 77 within nodules, 50

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day-old plants with mature nodules (inoculation with strain WT) were watered with

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different chemical treatments. Reactive N (10 mmol L-1 KNO3 and KNO2) was

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supplied to suppress N2 fixation, 22, 23 while 0.25 mmol L-1 Na2MoO4 (a readily

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soluble form of molybdate) was used to enhance nitrogenase activity. 24-26 Based on

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our preliminary experiments and previously published work, treatments with

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nitrogenase inhibitors or stimulants led to significant changes in nitrogenase activities

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within two to seven days. 23, 24, 26 Thus, plants were harvested five days after exposure

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to nitrogenase inhibitors or stimulant. PCB-supplemented seedlings treated with

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sterile distilled water were used as blank controls. Experiments were performed in

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triplicate.

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2.4. Nitrogen fixation measurements

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Nitrogenase activity of alfalfa nodules was measured using acetylene reduction assays

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(ARA). 27 Freshly harvested and intact nodulated roots were enclosed in 10 mL serum

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bottles filled with 10% acetylene and incubated at 25 °C. After 2 h, the amount of

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ethylene in the gas phase was quantified using an Agilent 6850 gas chromatograph

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equipped with a hydrogen flame ionization detector and a HP-PLOT U column (30 m

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× 0.32 mm, 10 µm) (Agilent Technologies, La Jolla, CA, USA). The injection and 9 / 27



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detector temperatures were 120 °C and 220 °C respectively. The column was held at

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50 °C for 8 min. The amount of ethylene produced was assessed by measuring the

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height of the ethylene peak on the chromatogram relative to the length of the reaction.

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Dilution of pure ethylene was employed to create five-point standard calibration

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curves (y = 15.668x + 48.094, R² = 0.998). The detection limit for ethylene was ~2

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nmol L-1. Ethylene production occurred linearly over the assay period.

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2.5. Net ion fluxes

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Net chloride ion (Cl−) and proton (H+) fluxes around the nodule surface were

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measured using the non-invasive micro-test technology (NMT100 Series,

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YoungerUSA LLC, Amherst, MA01002, USA) in the YoungerUSA NMT Service

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Center (Xuyue (Beijing) Sci. & Tech. Co., Ltd., Beijing, China). 28, 29 Preparation

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steps and calibration procedures for the ion microsensor are described in the

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Supplementary Text. 30 Briefly, 3-5 cm of freshly harvested nodulated roots

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(55-day-old) were dipped into corresponding measuring solution and equilibrated for

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10 min. Then, net ion flux of the nodules was monitored and steady-state flux was

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recorded for 10 min (Fig. S2). The micro-volt differences (DmV) were converted into

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net Cl− and H+ fluxes using JCal V3.3.2 (MS Excel spreadsheet,

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http://youngerusa.com). Negative and positive values represent influx and efflux,

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respectively.

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2.6. Hydrogen (H2) evolution 10 / 27


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The Q-Box NF1LP package (Qubit Systems Inc, Kingston, Canada) was used to

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measure the rate of H2 production in nodules (Fig. S3; Supplementary Text). 31 The

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nodulated roots were placed in a compartment, which was sealed with Qubitac

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Sealant to avoid air leakage. The volumes of H2 emitted (µmol g-1 nodule DM h-1)

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were measured using a H2 sensor (Model S121, Qubit) interfaced with a LabPro data

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logger (Vernier Software, Portland, Oregon). For each treatment, samples were

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collected from three different pots and a total of 15 plants were removed from each

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pot.

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2.7. PCB extraction and quantification

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Two grams of freeze-dried plant tissues (shoots, roots and nodules) were thoroughly

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homogenized, extracted twice with 60 ml hexane/acetone (1:1 v/v) overnight, and

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sonicated at 25°C for 15 min. 11, 17 The combined extracts were centrifuged and

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condensed to 5 mL using rotary evaporation. Each extract was then eluted with 25 mL

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of n-hexane through a purification filter comprising in sequence: silicon gel, Al2O3,

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acidified silicon gel, and anhydrous Na2SO4 powder (2:2:1:1, m/m). The eluate was

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concentrated and dissolved in 2 mL n-hexane. PCB 77 concentrations were quantified

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using an Agilent gas chromatograph (GC) equipped with an electron capture detector.

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A HP-5 column (30 m, 0.25-µm phase thickness, 0.32-mm inner diameter; J&W

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Scientific; Agilent Technologies) was used.

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To detect PCB 77 metabolites (e.g. biphenyl and methyl-biphenyls), gas 11 / 27



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chromatography-mass spectrometry (GC-MS) was performed using an Agilent 6890A

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GC system linked 5975C mass selective detector and a DB-5 MS column (30 m ×

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0.25 mm × 0.25 µm). 9, 11 Ultra-high performance liquid chromatography/tandem

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mass spectrometry (UPLC-MS/MS) was used to detect OH-PCB metabolites. 11

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Detailed parameters are listed in Supplementary Text. Note that one product was

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proposed to be a methoxy-trichlorobiphenyl compound based on the GC-MS

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spectrum of 4-methoxy-2,2',5'-trichlorobiphenyl, However, the exact compound

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formed could not be determined due to the unavailability of possible standards.

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2.8. Quantitative Real-Time PCR (qRT-PCR)

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Total RNA was extracted from frozen nodules using TRIzol reagent (Invitrogen,

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Carlsbad, CA) according to the manufacturer’s instructions. For each treatment,

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qRT-PCR experiments were performed with three independent RNA preparations,

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using the CFX96 Optical Real-Time Detection System (Bio-Rad) (details shown in

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Supplementary Text). Quantitative PCRs for nifH (encoding the Fe protein of

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nitrogenase) and legHb (encoding the LegHb) were performed using SYBR Premix

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Ex Taq system (Takara). rpsF and Smc-16S were used as reference genes for data

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normalization in S. meliloti NM (Fig. S4). mcs27 and actin2 were used as reference

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genes for normalisation in M. sativa. The nucleotide sequences of all primers used are

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listed in Table S2.

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2.9. Statistical analysis 12 / 27


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Experiments were performed in biological triplicate unless stated otherwise, and the

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data are expressed as the mean ± SD. Statistical significance was determined via a

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one-way analysis of variance 16 followed by Duncan's post-hoc test using SPSS 13.0

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software (SPSS Inc., Chicago, IL, USA). Differences in means were considered

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significant where p values < 0.05. A student's t-test was used for statistical

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comparisons of two means. Pearson correlation analysis was used to study the

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relationship between PCB dechlorination and other measured variables (ARA, H2

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production, H+ flux) with the SPSS, where Pearson’s correlation values (R) of 0.9 to

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1.0 demonstrate a near-total correlation and values from 0.7 to 0.9 indicate a strong

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correlation.

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3. Results and Discussion

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3.1 Nitrogenase activity and PCB transformation are strongly correlated in a

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rhizobial-legume symbiosis

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To study the impacts of N2 fixation on PCB transformation during symbiosis, nodular

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nitrogenase activity was manipulated using a series of chemical treatments. To

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determine the response of nitrogenase, nifH transcript levels and acetylene reduction

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activity (ARA) were measured five days after treatments were administered (Fig. 1A).

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The successful colonization of a GFP-tagged strain NM within PCB-fed alfalfa

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nodules was visualized by confocal microscopy (Fig. S5). Following inoculation of

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strain NM, mature pink nodules were produced (Fig. S1) and alfalfa growth was

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enhanced (Fig. S6). This suggests effective N2-fixing nodules were formed. 3 A slight 13 / 27



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but insignificant reduction in ARA was observed following PCB addition, while nifH

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expression was significantly reduced by 41.55% (p < 0.05). After exposure to MoO42-,

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N2-fixation capacity as measured by both expression level of nifH and nitrogenase

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activity increased sharply by 27- and 2.2-fold, respectively, compared to the control

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(WT + PCB) (Fig. 1A). This result is consistent with previous findings of rapid

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nitrogenase response to MoO42- in both symbiotic and free-living species 24, 25, 32 and

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reflects that Mo is part of the primary cofactor required for nitrogenase activity. 24-26

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Consistent with previous reports, 22, 23 addition of reactive nitrogen (NO2− and NO3−)

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significantly suppressed nitrogenase transcription and activity (p < 0.05), with 39-46%

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and 49-100% decreases below control levels (WT + PCB), respectively. To further

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manipulate nitrogenase levels, a mutant was constructed in the gene nifA, a

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transcriptional activator of nifHDK 33. In nodules inoculated with this mutant, plants

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exhibited a N2-fixation-deficient phenotype (Fig. S1), nifH expression was

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significantly down-regulated (p < 0.05) and nitrogenase activity was barely detectable

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(Fig. 1A). Thorough characterization of the transcription profile of nifHDK cluster

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would further help to illustrate the impacts of these treatments on nitrogenase activity.

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In combination, these data validate that NO3−, NO2−, and nifA deletion impedes, and

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MoO42− stimulates, nodular N2-fixation through transcriptional regulation.

280 281

PCB 77 dechlorination activity was measured in microaerobic PCB-supplemented

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nodules, in response to the same treatments, by quantifying net flux of Cl− ions (Fig.

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1B). PCB addition resulted in a marked increase in Cl− efflux (by 31%) in strain 14 / 27


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WT-inoculated nodules (P < 0.05) (Fig. S7), suggesting the compound was

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dechlorinated within the microoxic bacteroids. In line with N2 fixation activity, a

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7.2-fold increase in Cl− efflux was recorded following MoO42- treatment relative to

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controls (P < 0.05). In contrast, the average amount of Cl− efflux decreased 29%

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relative to controls (WT + PCB) following NO3− addition (P < 0.05) and changed

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only marginally following NO2− incubation (P > 0.05). In plants colonized with nifA

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mutants lacking nitrogenase activity, Cl− efflux was 72% lower than that of WT

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treatment (P < 0.05); however, no significant increase in Cl− release was observed

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after exposure to PCB (P > 0.05) in the mutant group, indicating that dechlorination

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may be weak in these conditions.

294 295

In combination, these findings suggest that the addition of Mo ions affect rates of

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transformation of PCB more strikingly than the repression conferred by NO3− and

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NO2−. It should be noted that Mo participates in a range of biochemical processes in

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addition to nitrogen fixation. Most notably, it serves as a cofactor in enzymes

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involved in the denitrification and nitrogen assimilation (i.e. nitrate reductase, NaR) 25

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and several studies suggest that NaR activity in crude microbial and plant extracts

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enhances the dechlorination of organohalides. 34, 35 However, contrary to these reports,

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our data show that supplemental NO3−, the substrate of NaR, significantly suppresses

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rhizobial dechlorination in intact plants. Furthermore, administration of the plant

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NaR-specific inhibitor tungstate (1 mM Na2WO4) 35 and inoculation with strain NarA

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mutant (which compromises the expression of the NaR catalytic subunit, napA) did 15 / 27



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not significantly decrease Cl− efflux (Fig. S7). This suggests that Mo stimulation of

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dechlorination rates is more related to nitrogenase activity than microbial or plant

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NaR activity. However, other explanations are also possible: Mo may modulate plant

309

uptake rates, serve as a cofactor in an as-yet-unidentified step in the PCB

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dechlorination pathway, or alternatively stimulate a bacterial co-metabolic process to

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facilitate PCB transformation.

312 313

3.2 Rhizobia dechlorinate PCB 77 to biphenyl and methyl-biphenyls within root

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nodules

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To further confirm whether nitrogenase activity was linked to higher rates of PCB

316

transformation, we measured residual PCB levels in different plant tissues (Fig. 2).

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The total contents of PCB 77 in strain WT-treated shoots and roots were 61% and 102%

318

higher than that in non-inoculated plants (P < 0.05), indicating rhizobial inoculation

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improved phytoextraction of PCB 77. 8 Consistent with previous reports, 13 nodules

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were prone to accumulate PCB 77, with levels in nodules higher than that in roots and

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shoots for all treatments (P < 0.05). The application of MoO42- dramatically reduced

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PCB 77 concentrations by 96% and 93% in alfalfa roots and nodules, respectively,

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compared to the control (WT + PCB) (P < 0.05) (Fig. 2A), indicating accelerated

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biotransformation of PCB 77 within plant belowground tissues. In contrast, the

325

contents of PCB 77 were only slightly affected by NO3- and NO2- treatment. The

326

mutant lacking N2-fixation ability poorly accumulated PCB in roots and nodules, with

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no dechlorinated intermediates detected in tissues, likely as a result of impaired root 16 / 27


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development (data not shown). There were no significant changes in PCB 77 levels of

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shoot biomass from all the groups (Fig. 2B), probably due to the relatively short

330

exposure periods of five days.

331 332

We subsequently used GC-MS and UPLC-MS/MS to detect PCB degradation

333

products under the different treatments. Various dechlorinated biphenyl products (i.e.

334

biphenyl and methyl-biphenyls) were detected by GC-MS in belowground parts of

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WT inoculated plants after MoO42- addition (Fig. 3, Fig. S8), but not in other

336

treatments. This is consistent with Mo addition accelerating rhizobial dechlorination

337

or degradation of PCB 77, possibly through enhanced nitrogenase activity. The

338

inability to detect biphenyl derivatives in the WT + PCB group may reflect low

339

transformation rates under this condition, making products hard to detect by GC-MS.

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Nevetheless, several congeners of hydroxyl-biphenyls (OH-BPs) were detected in

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below-ground parts of WT-inoculated alfalfa using UPLC-MS/MS (Fig. 3, Fig. S9),

342

further confirming that strain NM mediates PCB dechlorination. Further strategies

343

would be required to determine the exact structure of these metabolites in vivo, such

344

as nuclear magnetic resonance (NMR). In non-inoculated alfalfa, no putative

345

dechlorinated and hydroxylated intermediates of PCB 77 were detected after PCB

346

exposure. This is consistent with previous reports that, in contrast to some plant

347

species, 11, 14, 15 alfalfa cannot degrade polychlorinated compounds and its

348

transformation abilities are reported to be limited to hydroxylation of the

349

monochlorinated compound 4-chlorobiphenyl (Fig. S10). 16 In combination, these 17 / 27



350

data demonstrate that the bioconversion of PCB 77 is principally mediated by

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rhizobial dechlorination within nodules under these growth conditions. The mass

352

balance and uptake/transformation rates of PCB 77 and its metabolites within alfalfa

353

tissues under N2-fixing conditions bear further confirmation and investigation.

354 355

Our ongoing studies are now attempting to identify and characterize the key enzymes

356

mediating the transformation of PCB 77 to biphenyls within the microoxic nodules.

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The products identified by mass spectrometry suggest that legume-rhizobium

358

symbioses may degrade PCBs through novel reductive dehalogenation processes; the

359

dominant degradation products detected were methyl- and hydroxyl- substituted

360

biphenyls (Fig. S8, S9), in contrast to the methoxy-derivatives formed from oxidative

361

dechlorination processes reported in most plants. 11 This is in line with our previous

362

studies, which showed strain NM aerobically degrades tri- and tetra-chlorobiphenyls

363

to 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) and benzoates, 9, 17 rather

364

than the chloro-derivatives reported in most aerobes. 5, 16 Also consistently,

365

transcriptome sequencing shows that there are several dehalogenases (Rdases) found

366

in strain NM genome and one putative Rdase is upregulated by rhizobia during PCB

367

77 biodegradation in microoxic nodules (Table S4). However, to date the only

368

characterized RDases that can catalyze PCB dechlorination are three enzymes purified

369

from strictly anaerobic Dehalococcoides spp. 36 Several oxygen-tolerant RDases have

370

recently been purified from both anaerobic and aerobic bacteria that have been shown

371

to perform carbon–chlorine bond breakage under aerobic conditions. 37, 38 Hence, it is 18 / 27


Page 18 of 32

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372

plausible that S. meliloti may harbor similar enzymes to reductively degrade PCBs to

373

biphenyls in nodules.

374 375

3.3 Optimal dechlorination occurs in the microoxic environment of

376

nitrogen-fixing bacteroids

377

Given these findings, we hypothesized that the correlation between nitrogen fixation

378

and tetrachlorobiphenyl dechlorination activities may reflect that both activities

379

optimally occur in select microenvironments. Specifically, we hypothesized that a low

380

oxygen environment may be required for reductive dechlorination activity, similarly

381

to how such an environment is necessary to protect the oxygen-sensitive nitrogenase

382

enzyme. To test this, we measured the gene expression of plant LegHb, a marker for

383

nitrogenase function, which sequesters O2 in the cytoplasm to maintain a steady-state

384

of low O2 concentration within bacteroids. 3 We observed that PCB addition repressed

385

legHb expression, possibly by causing plant cellular redox imbalance, 39, 40 whereas

386

MoO42- reversed this effect and resulted in a 150-fold increase in expression (Fig. 4A).

387

The suppression of legHb expression by NO3- and NO2- was also noted; the supplied

388

N is believed to oxidize LegHb, resulting in the ferric form of this hemoprotein that is

389

unable control O2 levels within bacteroids. 23

390 391

We also hypothesized that nitrogenase-mediated H2 production may directly benefit

392

for dechlorination activity. Within bacteroids, N2 fixation is typically accompanied by

393

the evolution of H2 in an obligatory side reaction of nitrogenase. 31, 41 Consistently, we 19 / 27



394

observed that H2 evolution was modulated by NO3− and MoO42− addition (P < 0.05)

395

(Fig. 4B), but not NO2− exposure, and occurred in parallel with ARA and chloride

396

efflux (Table S3). In nifA mutant nodules, a twofold inhibition in H2 production was

397

observed, which likely reflects impaired N2 fixation in nodules. The rate of H+ import

398

was also observed to be partially correlated with ARA and dechlorination rates (Fig.

399

S11, Table S3). However, the mechanistic basis for this requires further investigation.

400 401

Although some rhizobia recycle endogenous H2 with an uptake hydrogenase (Hup+),

402

this enzyme is absent from the S. meliloti NM genome and the H2 generated instead

403

diffuses into the adjacent soil. 42 While traditionally viewed as a wasteful byproduct,

404

there is increasing evidence that nitrogenase-derived H2 is in fact beneficial for the

405

bacterium, host, and the wider rhizosphere, perhaps as an antioxidant or electron

406

donor. 43, 44 For example, within organochloride-contaminated ecosystems, it is

407

plausible that the H2 released may promote activity of hydrogenotrophic

408

dehalogenating consortia, 45 notably Dehalococcoides spp., that perform

409

dehalorespiration using H2 as an electron donor. 46 In our controlled pot experiments,

410

it is more likely that H2 promotes dechlorination through indirect mechanisms instead,

411

given no dehalogenating hydrogenotrophs are known to be present. It is possible that

412

increased H2 production helps to maintain a more reducing environment favorable for

413

dechlorination. In combination, these data show that optimal dechlorination occurs in

414

a microoxic environment and suggests that nitrogenase-mediated H2 evolution is

415

correlated with, and may even directly benefit, rhizobial dechlorination of PCB in 20 / 27


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416

nodules.

417 418

3.4 Implications, limitations and future directions

419

Using the alfalfa-rhizobium symbiosis as an example, we showed that the formation

420

of N2-fixing root nodules is required for phytoextraction of PCB under the growth

421

conditions tested. Our findings further demonstrated that biological dechlorination of

422

PCB 77 was linked to symbiotic N2 fixation. The expression and activity of

423

nitrogenase strongly correlated with the rates of Cl− efflux and PCB biotransformation.

424

Consistently, correlation analysis showed that the net efflux of Cl− was well correlated

425

to the ARA, nifH gene expression and H2 evolution (Fig. 5), with Pearson coefficients

426

of 0.836, 0.989, and 0.872 (p < 0.01) respectively (Table S3).

427 428

However, the functional basis of how nitrogenase activity is linked to PCB

429

dechlorination remains unclear. Ni et al. (2013) reported that nitrogenase itself can

430

directly dehalogenate difluorocyclopropene in vitro, 47 but it seems unlikely that

431

nitrogenase could act on compounds as large as PCB 77 at physiologically-significant

432

rates. Instead, it is more plausible that wider regulatory changes induced by the

433

formation of N2-fixing nodules and the maintenance of a microoxic environment

434

promote the uptake and transformation of PCBs. The oxygen-depleted, hydrogen-rich

435

atmosphere of the nodule may be particularly important to promote a suitably

436

reducing environment required for enzymatic reductive dechlorination. However, a

437

deeper understanding of the regulatory and biochemical aspects of these links depends 21 / 27



438

on the identification of the dehalogenases responsible for PCB 77 dechlorination in

439

rhizobia and the determination of whether RDases are responsible. 48 Future studies

440

are also required to precisely determine how Mo stimulates dechlorination and

441

whether H2 is beneficial for remediation. Furthermore, investigation of the coupling

442

effect on other widely distributed PCB congeners would be of practical significance

443

for in situ remediation strategies.

444 445

The knowledge gained from this study may be relevant for rehabilitating and

446

managing polluted sites. Our study suggests that in situ bioremediation of

447

organohalide-polluted sites may benefit from increased N2 fixation and that Mo is a

448

potentially crucial micronutrient limiting organohalide dechlorination. Hence, field

449

studies are now justified to determine whether planting leguminous plants and

450

administering molybdate fertilizer enhances bioremediation, especially under the

451

contaminant concentrations that occur in the environment. It is also notable that in situ

452

bioremediation of organohalide-polluted sites is often limited by N supply, 21 which

453

could be alleviated by biological N2 fixation. It should also be tested whether these

454

findings extend to free-living heterotrophic diazotrophs, which are of major

455

importance in both terrestrial and marine ecosystems. 32 Combined with previous

456

work on N2-fixing anaerobic dechlorinators, 21 our findings also highlight the

457

environmental roles played by nitrogenase and demonstrate the functional versatility

458

of legume-rhizobia symbioses beyond their natural role in N2 fixation.

459 22 / 27


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Page 23 of 32


460

ASSOCIATED CONTENT

461

Acknowledgements

462

This research was supported by the National Natural Science Foundation of China

463

(No. 41371309, 41671327 and 41230858), the Outstanding Youth Fund of Jiangsu

464

Province (No. BK20150049), and an ARC DECRA Fellowship (DE170100310;

465

awarded to C.G.).

466 467

Supporting Information Available

468

This supporting information is available free of charge via the Internet at

469

http://pubs.acs.org.

470

Eleven figures showing photographs of alfalfa growth (Figure S1), ion fluxes

471

measurement using microelectrodes (Figure S2), Q-Box NF1LP package for H2

472

measurement (Figure S3), gel electrophoresis for qPCR (Figure S4), colonization by

473

GFP-labeled strain NM (Figure S5), alfalfa growth parameters (Figure S6), net Cl−

474

efflux in nodules (Figure S7), GC-MS chromatograms of PCB 77 and its metabolites

475

(Figure S8), UPLC-MS/MS chromatograms of PCB 77 metabolites (Figure S9),

476

transformation of PCBs in different plant species (Figure S10), net H+ influx in

477

nodules (Figure S11). Detailed methodology (Supplementary Text).

478

Four tables listing information on strains and plasmids (Table S1), primers (Table S2),

479

Pearson`s correlation coefficients (Table S3), and transcriptome sequencing data for

480

Rdases (Table S4).

481 23 / 27



482

Author Contributions

483

Y.T., Y.M.L., X.M.W. and C.T. designed the research; X.M.W. conducted the

484

experiments. N.Z. and S.X.D. assisted in experimental work. Y.T, X.M.W. and C.G.

485

analyzed results and wrote the manuscript. W.J.R., L.Z., and Z.G.L. assessed and

486

commented on the manuscript.

487 488

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Figure 1. N2 fixation regulation and activity (A) and net Cl− flux (B) in nodules of alfalfa. After five days of PCB exposure, plants were treated with different chemicals (10 mmol L-1 KNO3, 10 mmol L-1 KNO2, and 0.25 mmol L-1 Na₂MoO₄) for another five days. WT represents alfalfa inoculated with S. meliloti NM wild-type; Smy represents alfalfa inoculated with S. meliloti NM nifA mutant. (A) Nitrogenase activity was measured by ARA. The expression level of nifH was normalized against two alfalfa reference genes. Bars with different lowercase letters were significantly different (P < 0.05). (B) Representative real-time plots of chloride ion (Cl−) fluxes of alfalfa nodules. 57x20mm (300 x 300 DPI)


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Figure 2. PCB 77 concentrations of alfalfa after varied treatments. (A) Roots and nodules. (B) Shoots. Within the same plant tissue, bars with different lowercase letters were significantly different (P < 0.05). 58x23mm (600 x 600 DPI)



Figure 3. Scheme of inferred PCB 77 biodegradation pathways of alfalfa inoculated with S. meliloti strain NM. All metabolites shown were detected by GC-MS or UPLC-MS/MS. The addition of MoO42− significantly stimulated the dechlorination of PCB 77. The pathways that result in the formation of the metabolites shown in dotted boxes are incompletely understood. 85x104mm (300 x 300 DPI)


Page 30 of 32

Page 31 of 32


Figure 4. The relative expression levels of leghemoglobin (legHb) (A) and hydrogen evolution (B) in nodules of alfalfa after varied treatments. (A) The relative expression levels were obtained using the 2-∆∆Ct method. Data are presented as mean values of relative gene expression ± SD (n = 3) and are normalised against the reference genes (mcs27 and actin2 for alfalfa). Bars with different lowercase letters were significantly different (P < 0.05) from the control (WT + PCB). 50x17mm (600 x 600 DPI)



Figure 5. Cl− efflux in alfalfa nodules as a function of relative nitrogenase acetylene reduction activity (ARA), across different treatments. ARA (ethylene µmol g-1 nodules FW h-1). 43x32mm (600 x 600 DPI)


Page 32 of 32

Coupling between nitrogen fixation and tetrachlorobiphenyl

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