Plant-Induced Changes to Rhizosphere pH Impact ... - ACS Publications

Subscriber access provided by UNIV OF DURHAM

Environmental Processes

Plant-induced Changes to Rhizosphere pH Impact Leaf Accumulation of Lamotrigine but not Carbamazepine Sara L Nason, Elizabeth Lianne Miller, K.G. Karthikeyan, and Joel A. Pedersen Environ. Sci. Technol. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.estlett.8b00246 • Publication Date (Web): 11 May 2018 Downloaded from http://pubs.acs.org on May 12, 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 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 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.

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 18

Environmental Science & Technology Letters

2

Plant-induced Changes to Rhizosphere pH Impact Leaf Accumulation of Lamotrigine but not Carbamazepine

3

Sara L. Nason†, Elizabeth L. Miller‡, K.G. Karthikeyan†,§, Joel A. Pedersen*,†,‡,ǁ

1

4 5

†

Environmental Chemistry and Technology Program, University of Wisconsin, Madison, WI 53706 ‡

§

6 7

Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, WI 53706

ǁ

Department of Biological Systems Engineering, University of Wisconsin, Madison, WI 53706

Departments of Soil Science, Civil & Environmental Engineering, and Chemistry, University of

8

Wisconsin, Madison, WI 53706

9

*Corresponding author: [email protected]

10

ABSTRACT. Many ionizable organic contaminants (IOCs) are present in treated wastewater

11

used to irrigate edible crops and accumulate in plants under field conditions. Phytoavailability of

12

IOCs with pKa values between 4 and 9 may be affected by the pH of the rhizosphere (the water

13

and soil within 2-3 mm of the root surface). Plants can alter rhizosphere pH by 2 to 3 units in

14

either direction in response to nutrient availability. The effects of plant modulation of

15

rhizosphere pH on IOC accumulation has not been previously reported. Here we provide direct

16

evidence that plant-driven changes in rhizosphere pH impact accumulation of an IOC in plant

17

leaves. Using a modified hydroponic system, we found that rhizosphere pH was higher by 1.5-

18

2.5 units when plants received only nitrate rather than a combination of nitrate and ammonium.

19

Plant-driven changes to rhizosphere pH altered accumulation of lamotrigine (pKa = 5.7) but not

20

carbamazepine, a non-ionizable contaminant. Lamotrigine accumulation in leaves correlated

21

strongly with the concentration of the neutral species available in porewater. We expect plant-

22

driven changes in rhizosphere pH to be important across a wide range of plant species and IOCs.

23

Consideration of plant modulation of rhizosphere pH may be necessary to accurately predict IOC

24

bioaccumulation.

1

ACS Paragon Plus Environment


25

INTRODUCTION

26

Conventional wastewater treatment processes do not completely remove many ionizable

27

organic contaminants (IOCs), including pharmaceuticals.1 Irrigation of food crops with

28

reclaimed wastewater and effluent-dominated water sources is common in arid regions

29

worldwide2 and is expected to grow as global climate warms, population increases, and demands

30

on freshwater sources rise. Crop plants can accumulate pharmaceuticals in edible tissues under

31

field conditions,3–5 prompting the need to evaluate the potential risks associated with effluent

32

irrigation of food crops.

33

Understanding the controls on IOC availability to plants may allow identification of

34

situations that lead to unacceptable accumulation in food crops. Current models have had limited

35

success predicting IOC uptake by crops.3 A variety of processes in the immediate vicinity of root

36

surfaces (the rhizosphere) can impact contaminant uptake by plants. Plants alter rhizosphere pH

37

to maintain electrochemical equilibrium as they take in nutrients. Root cells release H+ to the

38

rhizosphere as they take in cations (e.g., NH4+). Energy for anion (e.g. NO3–) uptake is provided

39

via co-transport of H+ from the rhizosphere into cells.6 These processes can raise or lower

40

rhizosphere pH by 2-3 units up to 2-3 mm from the root surface, resulting in rhizosphere pH

41

values of 4-8, depending on initial conditions.6–8 Speciation impacts IOC degradability,9 sorption

42

to soil particles10,11 and plant roots,11,12 uptake by roots,11–13 and movement through plants.11,12,14

43

Additionally, organic root exudates can alter sorption, stimulate microbial growth, and influence

44

the degradative capability of microorganisms.15–18 Changes in rhizosphere pH can affect copper,

45

uranium, cadmium, and inorganic nutrient phytoaccumulation from soils.6,19–22 Published studies

46

on IOC phytoaccumulation have invoked differences between rhizosphere and bulk soil pH to

2


Page 2 of 18

Page 3 of 18


47

explain uptake trends;23–25 however, direct demonstration of the impact of plant-induced changes

48

in rhizosphere pH on IOC phytoaccumulation has not been previously reported.

49

This study focuses on the ionizable phenyltriazine anticonvulsant lamotrigine (LTG,

50

conjugate acid pKa = 5.7)26 and the non-ionizable tricyclic anticonvulsant carbamazepine (CBZ),

51

which have been detected in reclaimed wastewater (concentrations up to 488 and 1,110 ng·L-1,

52

respectively)27,28 and accumulate in effluent-irrigated plants.23,25 Both LTG and a primary CBZ

53

metabolite, 10,11-epoxycarbamazepine (epCBZ), can accumulate in carrots irrigated with

54

reclaimed wastewater to levels exceeding the threshold of toxicological concern at normal

55

ingestion rates.23 This threshold serves as a conservative indicator of when additional study of

56

toxic effects is warranted.29,30

57

We examined the effects of plant-driven changes to rhizosphere pH on trends in LTG and

58

CBZ accumulation by wheat plants. We cultivated wheat with different forms of inorganic

59

nitrogen in a growth system designed to isolate the rhizosphere.19–21 We measured pH and

60

pharmaceutical concentrations in the model rhizosphere and related them to accumulation of

61

CBZ and LTG. We tested an ionizable and a non-ionizable contaminant to discriminate between

62

effects on accumulation due to changes in rhizosphere pH and those due to other potential

63

changes to the plants caused by the different nitrogen sources.

64

MATERIALS AND METHODS

65

Plant Growth. Durum wheat (Triticum durum) seeds were sterilized, soaked, and

66

germinated in a damp paper towel. After 2-5 days, sprouted seeds were transferred to growth

67

cells (Figure S1).20,31,32 Four seeds were placed inside each cell, and plants were cultured

68

hydroponically for 21 days, allowing the roots to grow into a mat against a layer of 30-µm nylon

69

mesh (Text S.1.3).20

3



Page 4 of 18

70

On day 22, each growth cell was transferred to a model rhizosphere setup, placing the mesh

71

containing the root mat against a thin layer (5.00 g) of ultrapure quartz sand. A strip of cellulose

72

filter paper wicked nutrient solution to the sand from a reservoir (replenished every 2 days). All

73

components were autoclaved or washed with methanol to minimize microbial growth. Sand and

74

paper were saturated with nutrient solution (containing 100 µg·L-1 LTG or CBZ) for the 8-day

75

exposure period. Half the rhizosphere setups received nutrient solution with nitrate as the sole

76

nitrogen source; the other half received a solution containing a 1:2 ammonium-to-nitrate molar

77

ratio (Text S.1.3). Nutrient solutions had equivalent ionic strength and were adjusted to pH 5.7.

78

Solution composition was chosen to induce differences in rhizosphere pH while minimizing

79

effects on plant growth. Each treatment (pharmaceutical + solution combination) was replicated

80

nine times; controls lacking pharmaceuticals or plants were conducted in triplicate. One replicate

81

represents a growth cell containing four plants. After 8 days, above-ground tissues (hereafter

82

leaves), roots, and sand were collected, frozen at –80 °C, freeze-dried, and stored at –80 °C until

83

extraction. Plant and sand masses were measured before and after freeze-drying.

84

Extraction

and

Liquid-Chromatography-Tandem-Mass-Spectrometry

(LC-

85

MS/MS). Freeze-dried sand from each replicate was rehydrated with 10 mM CaCl2 for pH

86

determination.33 Freeze-dried plant samples were ground, spiked with mass-labeled internal

87

standards, and subjected to Accelerated Solvent Extraction with methanol (10,300 kPa, 80 °C).

88

Extracts were evaporated to dryness and reconstituted in 4:1 water:acetonitrile with 0.1% acetic

89

acid. We measured LTG, CBZ, and the primary CBZ metabolites epCBZ and 10,11-trans-

90

dihydroxycarbamazepine (diOH-CBZ) in leaf and root extracts, sand rehydration solution, and

91

nutrient solution before and after a two-day replenishment cycle using LC-MS/MS (Agilent 1260

4


Page 5 of 18


92

HPLC, Waters Xterra MS C18 column, Agilent 6460 triple quadrupole mass spectrometer, ESI+

93

source) (Text S.1.4-S.1.7).

94

RESULTS AND DISCUSSION

95

Plant Modulation of Rhizosphere pH. Wheat plants altered porewater pH in the model

96

rhizospheres in response to the form of inorganic nitrogen provided (Figure 1). Nutrient solution

97

pH in the reservoirs was initially 5.7 ± 0.05 and differed by 99% LTG was

162

in the neutral form. This difference has been noted previously23,25,35 and may be caused by ion

163

trapping in root cell vacuoles.3,24,25 Lamotrigine speciation is dominated by LTG0 in root cell

164

cytosol (pH 7-7.4)11 and by LTG+ in vacuoles (pH 4-5.5).11 Thus, if LTG0 crosses the vacuole

165

membrane, a portion will remain trapped in the vacuole as LTG+, unable to be translocated

166

through the plant. We also cannot exclude the possibility that CBZ and LTG accumulation

167

differs due at least in part to differential metabolism in planta. This topic merits future

168

investigation.

169

Environmental Implications. We isolated the effect of nitrogen source-induced

170

rhizosphere pH changes on plant accumulation of an IOC, a process that we expect also occurs in

171

the field and may increase in importance if elevated atmospheric CO2 concentrations promote

172

root exudation of organic acids and shift plant assimilation preferences to reduced nitrogen

173

forms.36–39 We expect our results to serve as a basis for future experimental studies that

174

incorporate more of the complexity that exists in field scenarios. Discussion is therefore

175

warranted of how simplifications in our model system may result in differences from what

176

occurs in the field.

177

The model rhizosphere consisted of ultrapure quartz sand with low pH buffering capacity and

178

low sorption capacity for CBZ and LTG.24 Sorption had little impact on our findings, but pH-

179

dependent sorption processes could significantly influence contaminant availability to plants

180

grown in field soils. The magnitude and spatial extent of rhizosphere pH change depends on the

181

buffering capacity of the soil. Nonetheless, even in a soil with high buffering capacity due to

8


Page 8 of 18

Page 9 of 18


182

30% CaCO3 content, chickpeas changed pH by ~2 units within 1 mm of the root surface.6

183

Overall, we expect plant-driven changes in rhizosphere pH to be important under field conditions

184

for the accumulation of IOCs with pKa values between 4 and 9, although pH-dependent sorption

185

to soil constituents and high soil buffering capacity may impact relative trends in

186

phytoavailability. Using different initial pH and nutrient ratios could result in a higher or lower

187

rhizosphere pH range than observed in our study.

188

We used durum wheat, a species used in previous rhizosphere pH studies.20 Other species

189

known to alter rhizosphere pH in response to inorganic nitrogen availability include maize,

190

sorghum, chickpea, Norway spruce, white lupine, white clover, tomato, and rapeseed.8,19 Species

191

demonstrated to alter rhizosphere pH include graminaceous and non-graminaceous monocots,

192

dicots, and species with and without N2-fixing symbionts.8 We expect the effect demonstrated in

193

this study to be important across most crop species, although the magnitude of plant-driven

194

changes in rhizosphere pH is species-specific and can vary among cultivars.8

195

Rhizosphere microbiota vary among soils and plant species and can affect pH, nutrient

196

availability, and contaminant degradation. Their effect on contaminant phytoavailability and the

197

impact of contaminants on rhizosphere microbial communities have received little study.

198

Although our growth system was not sterile, we expect that any influence of microorganisms on

199

our results was small; we saw no evidence of microbial growth, and the observed pH shifts

200

resembled previously reported plant-induced rhizosphere pH changes.20,32 Rhizosphere

201

microbiota are a potential factor that could cause differences between our model system and field

202

conditions, even when the same process of nutrient-driven rhizosphere pH change occurs.

203

We suggest future studies on plant accumulation of IOCs report the main forms of nitrogen

204

and other nutrients supplied to the plants and consider rhizosphere pH. Differences in compound

9



205

speciation between the rhizosphere and bulk soil may hinder the ability to predict plant uptake of

206

IOCs. State-of-the-art models for organic contaminant uptake allow specification of soil

207

porewater pH.11 While knowledge of rhizosphere pH is required to account for the differential

208

contaminant uptake demonstrated in the present study, the underlying processes leading to

209

modulation of rhizosphere pH are not currently incorporated into such models. We suggest future

210

models explicitly incorporate rhizosphere processes, as has been done for a model to predict

211

uranium phytoaccumulation.22 Plants irrigated with reclaimed wastewater are exposed to and

212

accumulate a large variety of IOCs; testing all current and future IOCs is impractical. Accurate

213

prediction of IOC accumulation in plants may require detailed understanding of the impact of

214

rhizosphere processes on IOC availability.

215

ASSOCIATED CONTENT

216

Supporting information (SI) is available. SI contains methodological details and additional

217

results noted in the main text.

218

AUTHOR INFORMATION

219

Corresponding Author

220

* Phone: +1 (608) 263-4971. Email: [email protected].

221

ORCID

222

Sara Nason: 0000-0002-3866-6399

223

Liz Miller: 0000-0002-9711-2863

224

K.G. Karthikeyan: 0000-0002-2478-4941

225

Joel Pedersen: 0000-0002-3918-1860

10


Page 10 of 18

Page 11 of 18


226

Notes

227

The authors declare no competing financial interest.

228

ACKNOWLDEGEMENTS

229

We thank Prof. Christina Remucal and Dr. Curtis Hedman for use of instruments and laboratory

230

space, Troy Humphry for help with growth cell construction, and Rhea Ewing for help with

231

artwork. This work was funded by USDA-CSREES Hatch project WIS01647 and U.S.-Israel

232

Binational Agriculture Research & Development Fund grant US-4771-14R. J.A.P. acknowledges

233

support from the William A. Rothermel Bascom Professorship.

234

REFERENCES

235 236

(1)

Wang, J.; Wang, S. Removal of pharmaceuticals and personal care products (PPCPs) from wastewater: A review. J. Environ. Manage. 2016, 182, 620–640.

237 238

(2)

Sato, T.; Qadir, M.; Yamamoto, S.; Endo, T.; Zahoor, A. Global, regional, and country level need for data on wastewater generation, treatment, and use. Agric. Water Manag. 2013, 130, 1–13.

239 240

(3)

Miller, E. L.; Nason, S. L.; Karthikeyan, K.; Pedersen, J. A. Root uptake of pharmaceutical and personal care product ingredients. Environ. Sci. Technol. 2016, 50, 525–541.

241 242

(4)

Wu, X.; Dodgen, L. K.; Conkle, J. L.; Gan, J. Plant uptake of pharmaceutical and personal care products from recycled water and biosolids: a review. Sci. Total Environ. 2015, 536, 655–666.

243 244 245

(5)

Calderón-Preciado, D.; Matamoros, V.; Savé, R.; Muñoz, P.; Biel, C.; Bayona, J. M. Uptake of microcontaminants by crops irrigated with reclaimed water and groundwater under real field greenhouse conditions. Environ. Sci. Pollut. Res. Int. 2013, 20, 3629–3638.

246 247 248

(6)

Neumann, G.; Römheld, V. Rhizosphere Chemistry in Relation to Plant Nutrition. In Marschner’s Mineral Nutrition of Higher Plants: Third Edition; Marschner, P., Ed.; Academic Press: Waltham, MA, 2012; pp. 347–368.

249 250 251

(7)

Hinsinger, P.; Plassard, C.; Tang, C.; Jaillard, B. Origins of root-mediated pH changes in the rhizosphere and their responses to environmental constraints: a review. Plant Soil 2003, 248, 43– 59.

252 253

(8)

Marschner, H.; Römheld, V. Root-induced changes in the rhizosphere: Importance for the mineral nutrition of plants. Zeitschrift für Pflanzenernährung und Bodenkd. 1986, 149, 441–456.

254 255 256

(9)

Gulde, R.; Helbling, D. E.; Scheidegger, A.; Fenner, K. PH-dependent biotransformation of ionizable organic micropollutants in activated sludge. Environ. Sci. Technol. 2014, 48, 13760– 13768.

257

(10)

Droge, S. T. J. D.; Goss, K.-U. Development and evaluation of a new sorption model for organic

11



cations in soil: contributions from organic matter and clay minerals. Environ. Sci. Technol. 2013, 47, 14233–14241.

258 259 260 261 262

(11)

Trapp, S. Bioaccumulation of Polar and Ionizable Compounds in Plants. In Ecotoxicology Modeling; Devillers, J., Ed.; Emerging Topics in Ecotoxicology: Principles, Approaches and Perspectives 2; Springer Science+Business Media: New York, NY, 2009; pp. 299–353.

263 264

(12)

Inoue, J.; Chamberlain, K.; Bromilow, R. H. Physicochemical factors affecting the uptake by roots and translocation to shoots of amine bases in barley. Pestic. Sci. 1998, 54, 8–21.

265 266

(13)

Meychik, N. R.; Yermakov, I. P.; Prokoptseva, O. S. Diffusion of an organic cation into root cell walls. Biochem. 2003, 68, 760–771.

267 268

(14)

Hsu, F. C.; Kleier, D. A.; Melander, W. R. Phloem mobility of xenobiotics. Plant Physiol. 1988, 86, 811–816.

269 270

(15)

Lefevre, G. H.; Hozalski, R. M.; Novak, P. J. Root exudate enhanced contaminant desorption: An abiotic contribution to the rhizosphere effect. Environ. Sci. Technol. 2013, 47, 11545–11553.

271 272 273

(16)

Vergani, L.; Mapelli, F.; Zanardini, E.; Terzaghi, E.; Di Guardo, A.; Morosini, C.; Raspa, G.; Borin, S. Phyto-rhizoremediation of polychlorinated biphenyl contaminated soils: An outlook on plant-microbe beneficial interactions. Sci. Total Environ. 2017, 575, 1395–1406.

274 275 276 277

(17)

Joner, E. J.; Johansen, A.; Loibner, A. P.; Cruz, M. A. Dela; Szolar, O. H. J.; Portal, J. M.; Leyval, C. Rhizosphere effects on microbial community structure and dissipation and toxicity of polycyclic aromatic hydrocarbons (PAHs) in spiked soil. Environ. Sci. Technol. 2001, 35, 2773– 2777.

278 279 280

(18)

Chaudhry, Q.; Blom-Zandstra, M.; Gupta, S.; Joner, E. J. Utilising the synergy between plants and rhizosphere microorganisms to enhance breakdown of organic pollutants in the environment. Environ. Sci. Pollut. Res. 2005, 12, 34–48.

281 282 283

(19)

Chaignon, V.; Bedin, F.; Hinsinger, P. Copper bioavailability and rhizosphere pH changes as affected by nitrogen supply for tomato and oilseed rape cropped on an acidic and a calcareous soil. Plant Soil 2002, 243, 219–228.

284 285 286

(20)

Bravin, M. N.; Martí, A. L.; Clairotte, M.; Hinsinger, P. Rhizosphere alkalisation - a major driver of copper bioavailability over a broad pH range in an acidic, copper-contaminated soil. Plant Soil 2009, 318, 257–268.

287 288 289

(21)

Bravin, M. N.; Garnier, C.; Lenoble, V.; Gerard, F.; Dudal, Y.; Hinsinger, P. Root-induced changes in pH and dissolved organic matter binding capacity affect copper dynamic speciation in the rhizosphere. Geochim. Cosmochim. Acta 2012, 84, 256–268.

290 291

(22)

Boghi, A.; Roose, T.; Kirk, G. J. D. A model of uranium uptake by plant roots allowing for rootinduced changes in the soil. Environ. Sci. Technol. 2018, 52, 3536–3545.

292 293 294

(23)

Malchi, T.; Maor, Y.; Tadmor, G.; Shenker, M.; Chefetz, B. Irrigation of root vegetables with treated wastewater: evaluating uptake of pharmaceuticals and the associated human health risks. Environ. Sci. Technol. 2014, 48, 9325–9333.

295 296 297

(24)

Paz, A.; Tadmor, G.; Malchi, T.; Blotevogel, J.; Borch, T.; Polubesova, T.; Chefetz, B. Fate of carbamazepine, its metabolites, and lamotrigine in soils irrigated with reclaimed wastewater: Sorption, leaching and plant uptake. Chemosphere 2016, 160, 22–29.

298

(25)

Goldstein, M.; Shenker, M.; Chefetz, B. Insights into the uptake processes of wastewater-borne

12


Page 12 of 18

Page 13 of 18


pharmaceuticals by vegetables. Environ. Sci. Technol. 2014, 48, 5593–5600.

299 300 301 302

(26)

Young, R. B.; Chefetz, B.; Liu, A.; Desyaterik, Y.; Borch, T. Direct photodegradation of lamotrigine (an antiepileptic) in simulated sunlight – pH influenced rates and products. Environ. Sci. Process. Impacts 2014, 16, 848–857.

303 304 305

(27)

Ferrer, I.; Thurman, E. M. Identification of a new antidepressant and its glucuronide metabolite in water samples using liquid chromatography / quadrupole time-of-flight mass spectrometry. Anal. Chem. 2010, 82, 8161–8168.

306 307

(28)

Nikolaou, A.; Meric, S.; Fatta, D. Occurrence patterns of pharmaceuticals in water and wastewater environments. Anal. Bioanal. Chem. 2007, 387, 1225–1234.

308 309 310

(29)

Houeto, P.; Carton, A.; Guerbet, M.; Mauclaire, A. C.; Gatignol, C.; Lechat, P.; Masset, D. Assessment of the health risks related to the presence of drug residues in water for human consumption: Application to carbamazepine. Regul. Toxicol. Pharmacol. 2012, 62, 41–48.

311 312

(30)

Munro, I. C.; Renwick, A. G.; Danielewska-Nikiel, B. The Threshold of Toxicological Concern (TTC) in risk assessment. Toxicol. Lett. 2008, 180, 151–156.

313 314 315

(31)

Bravin, M. N.; Michaud, A. M.; Larabi, B.; Hinsinger, P. RHIZOtest: A plant-based biotest to account for rhizosphere processes when assessing copper bioavailability. Environ. Pollut. 2010, 158, 3330–3337.

316 317

(32)

Chaignon, V.; Hinsinger, P. Heavy metals in the environment: a biotest for evaluating copper bioavailability to plants in a contaminated soil. J. Environ. Qual. 2003, 32, 824–833.

318 319

(33)

Methods of Soil Analysis, Part 3 - Chemical Methods; Bartels, J. M., Ed.; Soil Science Society of America: Madison, WI, 1996.

320

(34)

Taiz, L.; Zeiger, E. Plant Physiology; 5th ed.; Sinauer Associates, Inc.: Sunderlan, MA, 2010.

321 322 323

(35)

Riemenschneider, C.; Al-Raggad, M.; Moeder, M.; Seiwert, B.; Salameh, E.; Reemtsma, T. Pharmaceuticals, their metabolites, and other polar pollutants in field-grown vegetables irrigated with treated municipal wastewater. J. Agric. Food Chem. 2016, 64, 5784–5792.

324 325

(36)

Keiluweit, M.; Bougoure, J. J.; Nico, P. S.; Pett-Ridge, J.; Weber, P. K.; Kleber, M. Mineral protection of soil carbon counteracted by root exudates. Nat. Clim. Chang. 2015, 5, 588–595.

326 327

(37)

Jackson, R. B.; Reynolds, H. L. Nitrate and ammonium uptake for single-and mixed-species communities grown at elevated CO2. Oecologia 1996, 105, 74–80.

328 329

(38)

Bloom, A. J.; Burger, M.; Asensio, J. S. R.; Cousins, A. B. Carbon dioxide enrichment inhibits nitrate assimilation in wheat and Arabidopsis. Science 2010, 328, 899–904.

330 331

(39)

Ma, Q.; Wang, J.; Sun, Y.; Yang, X.; Ma, J.; Li, T.; Wu, L. Elevated CO2 levels enhance the uptake and metabolism of organic nitrogen. Physiol. Plant. 2017, 4, 467–468.

332

13



B

pore water pH

8

7

Page 14 of 18

A

A C

6

5 NO₃⁻

NH₄⁺ + NO₃⁻

NO₃⁻

NH₄⁺ + NO₃⁻

plants

no plants

333 334

Figure 1. Wheat plants altered porewater pH in model rhizospheres in response to the form of nitrogen

335

provided. Plants raised porewater pH when supplied with nitrate as the sole nitrogen source. The pH in

336

the nutrient solution reservoirs at the end of each solution replenishment cycle varied from the initial

337

value by ≤ 0.1 pH unit. Letters indicate statistical significance based on a Wilcoxon/Kruskal-Wallis test

338

with Steel-Dwass post hoc analysis (p ≤ 0.05). Bars represent mean values; error bars indicate one

339

standard deviation (n = 21 for treatments with plants; n = 9 for treatments lacking plants).

14


Page 15 of 18


bioconcentration factor (L·kg-1)

180

Form of Nitrogen

150

Nitrate Ammonium + Nitrate

120 90

p = 3x10-4

60 30 0

LTG roots CBZ roots LTG leaves CBZ leaves

340 341

Figure 2. Bioconcentration factors for lamotrigine (LTG) and carbamazepine (CBZ) in the roots and

342

leaves of wheat plants supplied with the indicated nitrogen sources. Lamotrigine accumulation in the

343

leaves of plants provided with only nitrate exceeded that in plants receiving both ammonium and nitrate

344

by a factor exceeding two. Bioconcentration factors were calculated by dividing the concentration in the

345

plant roots or leaves by that in the porewater at the end of the exposure period. Porewater concentrations

346

of LTG and CBZ did not differ between nitrate and ammonium + nitrate treatments (Figure S3).

347

Concentrations measured in the plant tissue followed the same trends as the shown bioconcentration

348

factors. Welch’s t-test was used for pairwise comparison between treatments. Error bars indicate one

349

standard deviation.

15



350 351

Figure 3. Lamotrigine (LTG) accumulation in wheat leaves and roots correlated with the concentration of

352

the neutral LTG species in porewater (calculated via the Henderson-Hasselbalch equation). White and

353

yellow circles correspond to plants provided with nitrate as the sole nitrogen source. Red and black

354

triangles correspond to plants provided with ammonium + nitrate. The bottom line shows a linear

355

regression of the concentration of LTG in leaves against the concentration of neutral LTG in porewater

356

(R2 = 0.73). The slope is 0.015 ± 0.002 (p = 1.4 × 10-5), the y-intercept does not differ from zero (p =

357

0.31). The top line shows a linear regression of LTG concentration in plant roots against the concentration

358

of neutral LTG in porewater (R2 = 0.27). The slope is 0.023 ± 0.009 (p = 0.032), the y-intercept is 23 ± 2

359

(p = 9.7 × 10-8).

16


Page 16 of 18

Page 17 of 18

360


TOC Art

361

362

17



Page 18 of 18

LTG0 Nutrient Solution

Nitrate Only

CBZ

pH

Nitrate + Ammonium

pH

+ LTG


+H

Plant-Induced Changes to Rhizosphere pH Impact ... - ACS Publications

Recommend Documents