Coupling between Nitrogen Fixation and Tetrachlorobiphenyl

Subscriber access provided by READING UNIV

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

Environmental Science & Technology

1

Coupling between nitrogen fixation and tetrachlorobiphenyl dechlorination in a

2

rhizobium-legume symbiosis

3 4

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

5

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

6 7

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

8

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

9

2 Key Laboratory of Coastal Environmental Processes and Ecological Remediation,

10

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

11

264003, P.R. China

12

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

13

Australia

14

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

15 16

*Corresponding author:

17

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

18

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

19

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

1 / 27

ACS Paragon Plus Environment


20

ABSTRACT

21

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

22

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

23

and Sinorhizobium meliloti was used to explore the relationships between symbiotic

24

nitrogen fixation and transformation of tetrachlorobiphenyl PCB 77 within this

25

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

26

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

27

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

28

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

29

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

30

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

31

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

32

hydroxylbiphenyls, and trichlorobiphenyl derivatives) were detected within nodules

33

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

34

and leghemoglobin expression correlated positively with nodular dechlorination rates,

35

suggesting a more reducing environment promotes PCB dechlorination. Our findings

36

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

37

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

38

rhizobia to enhance phytoremediation of halogenated organic compounds.

2 / 27


Page 2 of 32

Page 3 of 32


39

Table of Contents (TOC)

40 41

3 / 27



42

1. Introduction

43

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

44

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

45

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

46

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

47

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

48

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

49

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

50

attracted interest for their capacity to remediate sites contaminated with organic

51

pollutants. Most notable is their capacity to biodegrade polychlorinated biphenyls

52

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

53

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

54

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

55

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

56

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

57

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

58

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

59 60

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

61

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

62

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

63

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


Page 4 of 32

Page 5 of 32


64

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

65

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

66

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

67

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

68

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

69

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

70

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

71

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

72

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

73

example by enhancing pollutant bioavailability for the rhizosphere and alleviating

74

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

75

equipped with appropriate biodegradative capabilities is necessary to optimize

76

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

77

augmentation with non-degradative Sinorhizobium strain contributed little to PCB

78

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

79

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

80

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

81

meliloti strain NM significantly promotes alfalfa performance and PCB removal from

82

soils in both laboratory and field settings. 8, 10

83 84

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

85

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



86

with plant metabolism. Considering the complex metabolism and intimate cooperation

87

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

88

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

89

4, 17

90

pollutant degradation would deepen our understanding of the underlying mechanisms

91

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

92

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

93

reductive dechlorination against chloroethylene, though the mechanistic basis for this

94

is unclear. 21

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

95 96

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

97

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

98

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

99

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

100

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

101

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

102

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

103

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

104

inactive.

105 106

2. Materials and Methods

107

2.1. Chemicals 6 / 27


Page 6 of 32

Page 7 of 32


108

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

109

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

110

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

111

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

112

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

113

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

114

purity unless stated otherwise.

115 116

2.2. Microorganisms

117

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

118

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

119

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

120

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

121

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

122

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

123

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

124

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

125

mating procedure (Supplementary Text).

126 127

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

128

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

129

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



130

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

131

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

132 133

2.3. Inoculation experiments

134

Inoculations were performed to investigate the dynamic interplay between rhizobial

135

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

136

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

137

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

138

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

139

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

140

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

141

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

142

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

143

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

144

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

145

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

146

80 µmol m−2 s−1.

147 148

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

149

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

150

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

151

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


Page 8 of 32

Page 9 of 32


152

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

153

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

154

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

155

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

156

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

157

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

158

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

159

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

160

our preliminary experiments and previously published work, treatments with

161

nitrogenase inhibitors or stimulants led to significant changes in nitrogenase activities

162

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

163

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

164

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

165

triplicate.

166 167

2.4. Nitrogen fixation measurements

168

Nitrogenase activity of alfalfa nodules was measured using acetylene reduction assays

169

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

170

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

171

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

172

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

173

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



174

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

175

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

176

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

177

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

178

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

179

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

180 181

2.5. Net ion fluxes

182

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

183

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

184

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

185

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

186

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

187

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

188

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

189

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

190

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

191

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

192

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

193

respectively.

194 195

2.6. Hydrogen (H2) evolution 10 / 27


Page 10 of 32

Page 11 of 32


196

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

197

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

198

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

199

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

200

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

201

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

202

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

203

pot.

204 205

2.7. PCB extraction and quantification

206

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

207

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

208

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

209

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

210

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

211

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

212

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

213

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

214

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

215

Scientific; Agilent Technologies) was used.

216 217

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



218

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

219

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

220

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

221

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

222

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

223

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

224

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

225

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

226 227

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

228

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

229

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

230

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

231

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

232

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

233

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

234

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

235

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

236

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

237

listed in Table S2.

238 239

2.9. Statistical analysis 12 / 27


Page 12 of 32

Page 13 of 32


240

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

241

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

242

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

243

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

244

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

245

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

246

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

247

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

248

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

249

correlation.

250 251

3. Results and Discussion

252

3.1 Nitrogenase activity and PCB transformation are strongly correlated in a

253

rhizobial-legume symbiosis

254

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

255

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

256

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

257

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

258

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

259

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

260

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

261

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



262

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

263

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

264

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

265

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

266

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

267

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

268

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

269

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

270

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

271

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

272

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

273

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

274

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

275

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

276

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

277

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

278

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

279

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

280 281

PCB 77 dechlorination activity was measured in microaerobic PCB-supplemented

282

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

283

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


Page 14 of 32

Page 15 of 32


284

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

285

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

286

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

287

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

288

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

289

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

290

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

291

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

292

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

293

may be weak in these conditions.

294 295

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

296

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

297

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

298

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

299

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

300

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

301

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

302

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

303

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

304

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

305

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



306

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

307

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

308

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

310

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

311

facilitate PCB transformation.

312 313

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

314

nodules

315

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).

317

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

319

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

320

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

321

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

322

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

323

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

324

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

327

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


Page 16 of 32

Page 17 of 32


328

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

329

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

335

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.

340

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

341

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

351

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.

357

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

Page 19 of 32


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


Page 20 of 32

Page 21 of 32


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


Page 22 of 32

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

References

489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518

1.

Herridge, D. F.; Peoples, M. B.; Boddey, R. M. Global inputs of biological nitrogen fixation in

agricultural systems. Plant Soil 2008, 311 (1-2), 1-18. 2.

Vitousek, P. M.; Menge, D. N.; Reed, S. C.; Cleveland, C. C. Biological nitrogen fixation: rates,

patterns and ecological controls in terrestrial ecosystems. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2013, 368 (1621), 20130119. 3.

Udvardi, M.; Poole, P. S. Transport and metabolism in legume-rhizobia symbioses. Annu. Rev.

Plant Biol. 2013, 64, 781-805. 4.

Teng, Y.; Wang, X.; Li, L.; Li, Z.; Luo, Y. Rhizobia and their bio-partners as novel drivers for

functional remediation in contaminated soils. Front. Plant Sci. 2015, 6 (32), 1-11. 5.

Van Aken, B.; Correa, P. A.; Schnoor, J. L. Phytoremediation of polychlorinated biphenyls: new

trends and promises. Environ. Sci. Technol. 2010, 44 (8), 2767-2776. 6.

Li, Y.; Harner, T.; Liu, L.; Zhang, Z.; Ren, N.; Jia, H.; Ma, J.; Sverko, E. Polychlorinated biphenyls in

global air and surface soil: distributions, air−soil exchange, and fracConaCon effect. Environ. Sci. Technol. 2009, 44 (8), 2784-2790. 7.

James, M. O., Polychlorinated biphenyls: metabolism and metabolites. In PCBs: Recent Advances

in Environmental Toxicology and Health Effects; Robertson, L. W.; Hansen, L. G., Eds.; The University Press of Kentucky: Kentucky 2001; Vol. 1, pp 35-46. 8.

Xu, L.; Teng, Y.; Li, Z. G.; Norton, J. M.; Luo, Y. M. Enhanced removal of polychlorinated biphenyls

from alfalfa rhizosphere soil in a field study-The impact of a rhizobial. Sci. Total Environ. 2010, 408 (5), 1007–1013. 9.

Tu, C.; Teng, Y.; Luo, Y.; Li, X.; Sun, X.; Li, Z.; Liu, W.; Christie, P. Potential for biodegradation of

polychlorinated biphenyls (PCBs) by Sinorhizobium meliloti. J. Hazard. Mater. 2011, 186 (2), 1438-1444. 10. Wang, X. M.; Teng, Y.; Zhang, N.; Christie, P.; Li, Z. G.; Luo, Y. M.; Wang, J. Rhizobial symbiosis alleviates polychlorinated biphenyls-induced systematic oxidative stress via brassinosteroids signaling in alfalfa. Sci. Total Environ. 2017, 592 (15), 68-77. 11. Sun, J.; Pan, L.; Su, Z.; Zhan, Y.; Zhu, L. Interconversion between methoxylated and hydroxylated polychlorinated biphenyls in rice plants: an important but overlooked metabolic pathway. Environ. Sci. Technol. 2016, 50 (7), 3668-3675. 12. Chekola, T.; Voughb, R. L.; Chaney, L. R. Phytoremediation of polychlorinated 24 / 27


Page 24 of 32

Page 25 of 32


519 520 521

biphenyl-contaminated soils: the rhizosphere effect. Environ. Int. 2004, 30 (6), 799-804.

522

14. Liu, J.; Hu, D.; Jiang, G.; Schnoor, J. L. In vivo biotransformation of 3, 3′, 4, 4′-tetrachlorobiphenyl

523 524 525 526 527 528 529 530

by whole plants−poplars and switchgrass. Environ. Sci. Technol. 2009, 43 (19), 7503-7509.

531

17. Wang, X.; Teng, Y.; Luo, Y.; Dick, R. P. Biodegradation of 3, 3′, 4, 4′-tetrachlorobiphenyl by

532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560

Sinorhizobium meliloti NM. Bioresour. Technol. 2016, 201, 261-268.

13. Teng, Y.; Sun, X.; Zhu, L.; Christie, P.; Luo, Y. Polychlorinated biphenyls in alfalfa:accumulation, sorption and speciation in different plant parts. Int. J. Phyt. 2017, 19 (8), 732-738.

15. Wang, S.; Zhang, S.; Huang, H.; Zhao, M.; Lv, J. Uptake, translocation and metabolism of polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) in maize (Zea mays L.). Chemosphere 2011, 85 (3), 379-385. 16. Mackova, M.; Barriault, D.; Francova, K.; Sylvestre, M.; Möder, M.; Vrchotova, B.; Lovecka, P.; Najmanová, J.; Demnerova, K.; Novakova, M.; Rezek, J.; Macek, T., Phytoremediation of polychlorinated biphenyls. In Phytoremediation and Rhizoremediation; Mackova, M.; Dowling, D.; Macek, T., Eds.; Springer: Dordrecht 2006; pp 152-153.

18. Mehmannavaza, R.; Prasher, S. O.; Ahmad, D. Rhizospheric effects of alfalfa on biotransformation of polychlorinated biphenyls in a contaminated soil augmented with Sinorhizobium meliloti. Process Biochem. 2002, 37 (9), 955-963. 19. Damaj, M.; Ahmad, D. Biodegradation of polychlorinated biphenyls by rhizobia: a novel finding. Biochem. Biophys. Res. Commun. 1996, 218 (3), 908-915. 20. Ahmad, D.; Mehmannavaz, R.; Damaj, M. Isolation and characterization of smbiotic N2-fixing Rhizobium meliloti from soils contaminated with aromatic and chloroaromatic hydrocarbons: PAHs and PCBs. Int. Biodeter. Biodegr. 1997, 39 (1), 33-43. 21. Ju, X.; Zhao, L.; Sun, B. Nitrogen fixation by reductively dechlorinating bacteria. Environ. Microbiol. 2007, 9 (4), 1078-83. 22. Drevon, J. J.; Alkama, N.; Bargaz, A.; Rodiño, A. P.; Sungthongwises, K.; Zaman-Allah, M., The legume–rhizobia symbiosis. In Grain Legumes; Antonio, M. D. R., Ed.; Springer: New York 2015; pp 267-290. 23. Trinchant, J. C.; Rigaud, J. Nitrite and nitric oxide as inhibitors of nitrogenase from soybean bacteroids. Appl. Environ. Microbiol. 1982, 44 (6), 1385-1388. 24. Kaiser, B. N.; Gridley, K. L.; Ngaire Brady, J.; Phillips, T.; Tyerman, S. D. The role of molybdenum in agricultural plant production. Ann. Bot. 2005, 96 (5), 745-754. 25. Presta, L.; Fondi, M.; Emiliani, G.; Fani, R., Nitrogen fixation, a molybdenum-requiring process. In Molybdenum Cofactors and Their role in the Evolution of Metabolic Pathways; Presta, L.; Fondi, M.; Emiliani, G.; Fani, R., Eds.; Springer: Dordrecht 2015; pp 53-66. 26. Tejada-Jiménez, M.; Gil-Díez, P.; León-Mediavilla, J.; Wen, J. Q.; Mysore, K. S.; Imperial, J.; González-Guerrero, M. Medicago truncatula molybdate transporter type 1 (MtMOT1. 3) is a plasma membrane molybdenum transporter required for nitrogenase activity in root nodules under molybdenum deficiency. New Phytol. 2017, 216 (4), 1223-1235. 27. Hardy, R. W.; Holsten, R. D.; Jackson, E. K.; Burns, R. C. The acetylene-ethylene assay for N2 fixation: laboratory and field evaluation. Plant Physiol. 1968, 43 (8), 1185-1207. 28. Zeng, G. M.; Chen, A. W.; Chen, G. Q.; Hu, X. J.; Guan, S.; Shang, C.; Lu, L. H.; Zou, Z. J. Responses of Phanerochaete chrysosporium to toxic pollutants: physiological flux, oxidative stress, and 25 / 27



561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604

Page 26 of 32

detoxification. Environ. Sci. Technol. 2012, 46 (14), 7818-7825. 29. Ma, J.; Cai, H.; He, C.; Zhang, W.; Wang, L. A hemicellulose-bound form of silicon inhibits cadmium ion uptake in rice (Oryza sativa) cells. New Phytol. 2015, 206 (3), 1063-1074. 30. Yan, S.; McLamore, E. S.; Dong, S.; Gao, H.; Taguchi, M.; Wang, N.; Zhang, T.; Su, X.; Shen, Y. The role of plasma membrane H+-ATPase in jasmonate-induced ion fluxes and stomatal closure in Arabidopsis thaliana. Plant J. 2015, 83 (4), 638-649. 31. Peoples, M. B.; McLennan, P. D.; Brockwell, J. Hydrogen emission from nodulated soybeans [Glycine max (L.) Merr.] and consequences for the productivity of a subsequent maize (Zea mays L.) crop. Plant Soil 2008, 307 (1-2), 67-82. 32. Barron, A. R.; Wurzburger, N.; Bellenger, J. P.; Wright, S. J.; Kraepiel, A. M. L.; Hedin, L. O. Molybdenum limitation of asymbiotic nitrogen fixation in tropical forest soils. Nat. Geosci. 2009, 2 (1), 42-45. 33. Rubio, L. M.; Ludden, P. W. Biosynthesis of the iron-molybdenum cofactor of nitrogenase. Annu. Rev. Microbiol. 2008, 62, 93-111. 34. Magee, K. D.; Michael, A.; Ullah, H.; Dutta, S. K. Dechlorination of PCB in the presence of plant nitrate reductase. Environ. Toxicol. Pharmacol. 2008, 25 (2), 144-147. 35. De, S.; Perkins, M.; Dutta, S. K. Nitrate reductase gene involvement in hexachlorobiphenyl dechlorination by Phanerochaete chrysosporium. J. Hazard. Mater. 2006, 135 (1-3), 350-354. 36. Wang, S.; Chng, K. R.; Wilm, A.; Zhao, S.; Yang, K. L.; Nagarajan, N.; He, J. Genomic characterization of three unique Dehalococcoides that respire on persistent polychlorinated biphenyls. P. Natl. Acad. Sci. USA 2014, 111 (33), 12103-12108. 37. Chen, K.; Huang, L.; Xu, C.; Liu, X.; He, J.; Zinder, S. H.; Li, S.; Jiang, J. Molecular characterization of the enzymes involved in the degradation of a brominated aromatic herbicide. Mol. Microbiol. 2013, 89 (6), 1121-39. 38. Payne, K. A.; Quezada, C. P.; Fisher, K.; Dunstan, M. S.; Collins, F. A.; Sjuts, H.; Levy, C.; Hay, S.; Rigby, S. E.; Leys, D. Reductive dehalogenase structure suggests a mechanism for B12-dependent dehalogenation. Nature 2015, 517 (7535), 513-516. 39. Chang, C.; Damiani, I.; Puppo, A.; Frendo, P. Redox changes during the legume-rhizobium symbiosis. Mol. Plant 2009, 2 (3), 370-377. 40. Marino, D.; Damiani, I.; Gucciardo, S.; Mijangos, I.; Pauly, N.; Puppo, A. Inhibition of nitrogen fixation in symbiotic Medicago truncatula upon Cd exposure is a local process involving leghemoglobin. J. Exp. Bot. 2013, 64 (18), 5651–5660. 41. Milton, R. D.; Abdellaoui, S.; Khadka, N.; Dean, D. R.; Leech, D.; Seefeldt, L. C.; Minteer, S. D. Nitrogenase bioelectrocatalysis: heterogeneous ammonia and hydrogen production by MoFe protein. Energy Environ. Sci. 2016, 9 (8), 2550-2554. 42. Vignais, P. M.; Billoud, B. Occurrence, classification, and biological function of hydrogenases: an overview. Chem. Rev. 2007, 107 (10), 4206-4272. -

43. Favre, J. S. L.; Focht, D. D. Conservation in soil of H2 liberated from N2 fixation by Hup nodules. Appl. Environ. Microbiol. 1983, 46 (2), 304-311. 44. Jin, Q.; Zhu, K.; Cui, W.; Xie, Y.; Han, B.; Shen, W. Hydrogen gas acts as a novel bioactive molecule in enhancing plant tolerance to paraquat-induced oxidative stress via the modulation of heme oxygenase-1 signalling system. Plant Cell Environ. 2013, 36 (5), 956-569. 45. Marshall, I. P.; Berggren, D. R.; Azizian, M. F.; Burow, L. C.; Semprini, L.; Spormann, A. M. The Hydrogenase Chip: a tiling oligonucleotide DNA microarray technique for characterizing 26 / 27


Page 27 of 32


605 606 607 608 609 610 611 612 613 614

hydrogen-producing and -consuming microbes in microbial communities. ISME J. 2012, 6 (4), 814-826. 46. Men, Y.; Feil, H.; Verberkmoes, N. C.; Shah, M. B.; Johnson, D. R.; Lee, P. K.; West, K. A.; Zinder, S. H.; Andersen, G. L.; Alvarez-Cohen, L. Sustainable syntrophic growth of Dehalococcoides ethenogenes strain 195 with Desulfovibrio vulgaris Hildenborough and Methanobacterium congolense: global transcriptomic and proteomic analyses. ISME J. 2012, 6 (2), 410-421. 47. Ni, F.; Lee, C. C.; Hwang, C. S.; Hu, Y.; Ribbe, M. W.; McKenna, C. E. Reduction of fluorinated cyclopropene by nitrogenase. J. Am. Chem. Soc. 2013, 135 (28), 10346-10352. 48. van Pée, K. H.; Unversucht, S. Biological dehalogenation and halogenation reactions. Chemosphere 2003, 52 (2), 299-312.

615

27 / 27



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)


Page 28 of 32

Page 29 of 32


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

Recommend Documents