Toxicogenomic Responses of the Model Legume Medicago truncatula

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Toxicogenomic responses of the model legume Medicago truncatula to aged biosolids containing a mixture of nanomaterials (TiO2, Ag and ZnO) from a pilot wastewater treatment plant Chun Chen, Jason M Unrine, Jonathan D Judy, Ricky W. Lewis, Jing Guo, David H. Mcnear, and Olga V. Tsyusko Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b01211 • Publication Date (Web): 11 Jun 2015 Downloaded from http://pubs.acs.org on June 15, 2015

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Environmental Science & Technology

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Toxicogenomic responses of the model legume Medicago truncatula

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to aged biosolids containing a mixture of nanomaterials (TiO2, Ag

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and ZnO) from a pilot wastewater treatment plant

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Chun Chen1,2,3*, Jason M. Unrine1,2,3, Jonathan D. Judy1,2,3,4, Ricky W. Lewis1,2, Jing Guo5,

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David H. McNear Jr.1,2, Olga V. Tsyusko1,2,3*

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1

Department of Plant and Soil Sciences, University of Kentucky, Lexington KY, 40546, USA

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2

Transatlantic Initiative for Nanotechnology and the Environment (TINE)

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3

Center for the Environmental Implications for Nanotechnology (CEINT), Duke University, Durham, NC 27708,

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USA

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4

CSIRO Land and Water, Waite Campus, Urrbrae, South Australia, 5064, Australia

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5

Departments of Epidemiology and Biostatistics, University of Kentucky, Lexington KY, 40536, USA

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*

To whom correspondence may be addressed

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Chun Chen

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University of Kentucky

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Lexington, KY, 40504

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859-257-1978

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[email protected]

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19 20

Olga V. Tsyusko

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University of Kentucky

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Lexington, KY, 40504

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859-257-1777

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[email protected]

25 26 27 28 29 30 31 1 ACS Paragon Plus Environment


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ABSTRACT

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The toxicogenomic responses in Medicago truncatula A17 were monitored following exposure

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to biosolids-amended soils. Treatments included biosolids produced using a pilot wastewater

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treatment plant with either no metal introduced into the influent (control), bulk/ionic TiO2, ZnO,

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and AgNO3 added to influent (bulk/dissolved treatment), or Ag, ZnO, and TiO2 engineered

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nanomaterials added to influent (ENM treatment) and then added to soil which was aged in the

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field for six months. In our companion study we found inhibition of nodulation in the ENM but

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not in the bulk/dissolved treatment. Gene expression profiling revealed highly distinct profiles

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with more than 10-fold down-regulation in 239 genes in M. truncatula roots from the ENM

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treatment, while gene expression patterns were similar between bulk/dissolved and control

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treatments. In response to ENM exposure, many of the identified biological pathways, gene

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ontologies, and individual genes are associated with nitrogen metabolism, nodulation, metal

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homeostasis and stress responses. Expression levels of 9 genes were independently confirmed

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with qRT-PCR. Exposure to ENMs induced unique shifts in expression profiles and biological

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pathways compared with bulk/dissolved treatment, despite the lack of difference in bioavailable

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metal fractions, metal oxidation state and coordination environment between ENM and

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bulk/dissolved biosolids. As populations of Sinorhizobium meliloti Rm2011 were similar in

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bulk/dissolved and ENM treatments, our results suggest that inhibition of nodulation in the ENM

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treatment was primarily due to phytotoxicity, likely caused by enhanced bioavailability of Zn

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ions.

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INTRODUCTION

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The field of nanotechnology is developing rapidly and engineered nanomaterials (ENMs) are

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being incorporated into an increasing number of industrial and consumer products. The ENMs

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within these products are being released into the environment, which raises concerns about their

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possible impacts on human and ecosystem health.1 Metal/metal oxide nanoparticles (NPs), such

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as silver, zinc oxide (ZnO), and titanium dioxide (TiO2), are among the most widely used classes

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of ENMs. Due to the unique antibacterial properties of Ag, AgNPs are one of the most widely

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used ENMs in various consumer products and medical equipment.2, 3 ZnO and TiO2 NPs are UV

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filters, antibacterial agents, and corrosion inhibitors, hence they are increasingly being integrated

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into a wide range of industrial and consumer product (e.g. sunscreens, coatings, paints and

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photocatalysts).4-6

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Previous investigations have predicted that the majority of ZnO, TiO2 and Ag NPs from

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consumer products will end up in wastewater treatment plants (WWTP) where they will partition

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to sewage sludge during wastewater treatment,5,

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through the application of biosolids to agricultural soils or from other methods of disposal for

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biosolids (e.g. landfilling and incineration).8 Thus, soil may serve as a primary sink for ENMs

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accumulation in the environment, in which NPs may enter food webs or cause direct toxicity to

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plants, microbial communities, or other soil organisms.

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and ultimately re-enter the environment

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In the past few years, it has become clear that ENMs will be transformed during wastewater

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treatment and the aged ENMs (a-ENMs) discharged into terrestrial ecosystems will have

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fundamentally different physical and chemical properties than pristine ENMs.9-12 However, the

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vast majority of studies to date have examined the phytotoxicity of pristine or as-manufactured

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Ag, TiO2, or ZnO NPs, in hydroponic systems or in culture media. As a result, little information

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is available regarding the phytotoxicity of a-ENMs in soil.13

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Currently, little is known about how the accumulation of ENMs in soils will interfere with

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important plant-microbial relationships such as those that occur in legumes or for other plant

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growth promoting rhizobacteria (PGPR).

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symbiosis is critical for soil fertility and agricultural productivity.14 Recently, Priester et al.

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added manufactured NPs directly into organic farm soil and showed that ZnO NPs slightly

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stimulated plant growth, while exposure to a greater concentrations of CeO2 NPs (greater than

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500 mg kg-1) significantly inhibited nodule-associated N2 fixation.15

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focused on addition of pristine ENMs to soils rather than ENMs which had undergone

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wastewater treatment followed by aging (a-ENM). Hydroponic studies with Pisum sativum L.

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found that the presence of ZnO and TiO2 NPs at concentrations greater than 250 mg L-1 affect

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the early plant-rhizobia interactions, interfering with nodule development and subsequently

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delaying the onset of nitrogen fixation.16, 17 To our knowledge, no studies exist examining the

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effects of a-ENMs on rhizobium-legume symbiosis.

Nitrogen fixation resulting from rhizobia-legume

However, their study

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In this study, we exposed Medicago truncatula A17, an important forage crop and model

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legume, to soils that were amended with biosolids generated by a pilot waste water treatment

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plant (WWTP) and inoculated with Sinorhizobium meliloti Rm2011. Treatments consisted of

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biosolids generated with either no metal added to the WWTP influent (control), bulk/ionic ZnO,

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TiO2 and AgNO3 added to the influent (bulk/dissolved treatment), or Ag, ZnO and TiO2 ENMs

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added to the influent (ENM treatment). The companion paper published in this issue describes

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inhibition of soil microbial communities, plant growth and development and root nodulation, as

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well as increased metal accumulation, in the ENM treatment compared to the bulk/dissolved


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treatment.18 To determine whether observed inhibition of nodulation arose primarily from plant

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or microbial toxicity and to explore the mechanisms involved in phytotoxicity induced by the

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ENM exposure, here we implemented whole genome microarray analyses of M. truncatula (from

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shoot and root tissues) exposed to ENM and bulk/dissolved metals in soils amended with

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biosolids.

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MATERIALS AND METHODS

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Aged biosolids amended soil

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Details of the pilot scale WWTP, characterization of the nanomaterials used, the characteristics

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of the resulting biosolids have been described previously.11, 18

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designed to generate a large quantity of biosolids with Zn concentrations near the regulatory

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limits for Zn (2,800 mg Zn kg-1 dry mass) for long-term application of biosolids19, as well as Ag

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concentrations near the 98th percentile (180 mg Ag kg-1 dry mass), and Ti concentrations (5,000

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mg Ti kg-1 dry mass) near the maximum concentrations detected in the U.S. Targeted National

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Sewage Sludge Survey.20 Of the three metals, only Zn is regulated in biosolids in the US.19

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These concentrations were selected to represent a worst case scenario with land application for

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10 years. The biosolids were combined in a 0.58:0.42 ratio with a sandy soil. This ratio closely

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follows the assumption of a 1:1 ratio of soil to biosolids in the top 15 cm of soil for chronic

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laoading described in the Biosolids Risk Assessments for the EPA, Part 503 document.19 The soil

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biosolid mix was then aged for six months in outdoor lysimeters at Rothamstead Research in

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Harpenden, UK. Details of the soil and biosolid amended soil characterization were presented in

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the companion manuscript.18 Metal speciation of Zn and Ag as determined by X-ray absorption

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near edge structure (XANES) spectroscopy was similar in control, bulk/dissolved and ENM

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biosolids, as well as in the aged amended soil.11, 18

The pilot scale WWTP was



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Medicago truncatula exposures

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Wild-type A17 M. truncatula seeds were scarified with concentrated H2SO4 for 7 min and

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rinsed 5 times with sterile 18 MΩ deionized water (DI), and then surface sterilized with

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commercial bleach for 3 min and washed in DI water.21 Seeds were spread out in sterile Petri

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dishes and germinated at 25 ºC in the dark overnight. Six germinated seedlings with straight

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radicles (1-2 cm in length) were transferred to each circular pot (8×8×7 cm, 400 mL volume),

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containing about 340 g of amended soil prepared as described above. Each plant was inoculated

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with 1 mL of a washed suspension of S. meliloti in sterile DI water (OD600 = 0.8). Twelve pots

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in each treatment were randomly placed in a plant growth chamber with a 14 h light/10 h dark

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photoperiod at 20 ºC and 70% relative humidity.

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subsequently harvested, divided into shoots and roots, flash frozen in liquid nitrogen, and stored

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at -80 ºC prior to RNA extractions.

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Microarray Analyses

Plants were grown for 28 days and

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RNA extractions were conducted on the pooled shoots of 3 individuals per pot and the pooled

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roots of 6 individuals per pot. Three replicate pots were randomly selected from each treatment

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for extraction. Total RNA was extracted using the RNeasy® Plant Mini Kit (Qiagen, Hilden,

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Germany), and purified with DNase I (Qiagen) according to the manufacturer’s protocol. RNA

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was quantified and evaluated for purity using a Varian Cary 50 UV/Visible spectrophotometer

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(Agilent, Santa Clara, CA, USA) and Bioanalyzer (Agilent).

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integrity values (RIN 8~10) and were submitted to the microarray core facility at the University

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of Kentucky.

All RNA samples had high

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A total of 100 ng of RNA was used for all hybridizations to the Medicago truncatula

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Affymetrix GeneChip® (Santa Clara, CA, USA) containing 61,200 probe sets. cRNA probe


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labeling, amplification and hybridization were performed following the standard GeneChip

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expression analysis technical manual. GeneChips were washed and stained in the Affymetrix

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Fluidics Station 450. Arrays were scanned using an Affymetrix GeneChip Scanner 3000 7G.

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Raw expression data (Affymetrix CEL files) were normalized with the RMA (robust multiarray

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average)22 algorithm using Partek Genomics Suite version 6.6 (Partek Inc., St. Lousis, MO,

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USA). For multiple probes targeting the same genes and showing similar expression levels, we

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used the probe with the highest fold change in ENM or bulk/dissolved treatment compared to

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control. A one-way analysis of variance (ANOVA) with orthogonal contrasts (ENM vs control

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and bulk/dissolved vs control) was used to test for significance of differentially expressed genes

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(DEGs). DEGs that were up- or down-regulated by more than 1.5 fold at P < 0.05 without false

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discovery rate (FDR) adjustment were considered statistically significant. Identification of DEGs

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was also performed after FDR of 0.05 and 0.1 was applied (SI File S4). The FDR of 0.05 and 0.1

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produced 88 and 205 DEGs, respectively, in response to ENM treatment in roots. For shoots,

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only seven genes were identified at FDR of 0.1 for ENM and no DEGs were detected for

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bulk/dissolved treatment. However, for the pathway and GO analyses we have chosen to include

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all DEGs with unadjusted p-value. In order to control for the number of false positives,

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application of stringent FDR-criteria may lead to a high number of false negatives potentially

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increasing the type II error and resulting in elimination of biologically significant genes

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responding to a treatment.23-25 Raw microarray data from this study were deposited in the

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National Center for Biotechnology Information (NCBI)’s Gene Expression Omnibus database

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with the accession number “GSE64788”. Hierarchical clustering was performed in Partek for

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shoot and root samples using all DEGs (596 for shoots and 1128 for roots) to confirm that they

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match their treatment groups and to examine the grouping of control, bulk/dissolved and ENM



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treatments. The common and unique DEGs between bulk/dissolved and ENM treatments for

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shoots and roots were determined and visualized using Venn diagrams.

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In order to explore the biological significance of DEGs responsive to ENM and bulk/dissolved

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exposure, gene ontology (GO) categories analyses were performed by AgriGO using the

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Singular Enrichment Analysis tool according to a χ2 statistical test and the Yekutieli multi-test

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adjustment method.26 Significantly enriched GO terms were determined at P < 0.05, FDR < 0.05

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with a hypergeometric test. KEGG (Kyoto Encyclopedia of Genes and Genomes)27, 28 pathway

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functional enrichment analysis was performed using the pathway analysis module in Partek

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Genomics Suite. The significant KEGG pathways were identified with the criteria that pathways

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must have three or more DEGs with fold change > 1.5 and the DEGs in the pathway are

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overrepresented based on a hypergeometric test with P < 0.05. PathExpress29 was also used to

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identify the most relevant metabolic pathways associated with the same lists of DEGs. The

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default threshold of P < 0.05 with FDR adjustment was used for PathExpress analysis of

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overrepresented pathways.

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Quantitative Real-Time PCR (qRT-PCR) Validation

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Validation of microarray data was conducted via an independent qRT-PCR experiment. For

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shoots, we selected four significantly differentially regulated genes with respect to their relevant

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metabolic pathways that are significantly overrepresented identified by ENM exposure using

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PathExpress tool, as well as these candidate genes that have high fold changes. IFR (isoflavone

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reductase) and F3'H (flavonoid 3'-hydroxylase) are involved in isoflavonoid biosynthesis and

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flavonoid biosynthesis pathways, respectively. Both GST (glutathione S-transferase) and P450

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(cytochrome P450 71B10) are linked to pathway of xenobiotic or drug metabolism by

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cytochrome P450. For roots, five target genes including MTP (metal tolerance proteins), MTR

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(metal transporter), PEROX (peroxidase), NADPH (NADPH oxidase) and ACC_Oxidase (18 ACS Paragon Plus Environment

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aminocyclopropane-1-carboxylate oxidase-like protein) were selected from the top ten up-

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regulated DEGs in the ENM treatment (Table 1). Actin 2 was selected as a reference gene for

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relative quantification because it showed stable expression among treatments in the microarray

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data as confirmed with qRT-PCR (supporting information, Figure S1). For each of these genes,

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individual shoots (n = 5) and pooled root samples (n = 3) from each treatment were used for

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qRT-PCR analysis (See supporting information for the detailed methods). Primer sequences,

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probe sequences, amplicon sizes and amplification efficiencies are listed in the Table S1.

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RESULTS AND DISCUSSION

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Overall, the microarray data from M. truncatula exposed to ENM and bulk/dissolved

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treatments relative to the control showed that a significantly higher number of DEGs (570 vs 79

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in shoots and 1114 vs 21 in roots) (Figure 1B), associated with more KEGG pathways (6 vs 2 in

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shoots and 12 vs 0 in roots) (Table 2) and GOs (55 vs 0 in shoots and 118 vs 17 in roots)

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(supporting information, File S2) were observed in the ENM than the bulk/dissolved treatment.

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In roots from the ENM exposure, 239 genes were down-regulated more than 10-fold, with some

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of the genes from the nodulin family showing down-regulation at more than 1000 fold (Figure

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1C). A complete lists of the identified DEGs, GOs and pathways are presented in supporting

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information (File S1, S2 and S3).

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According to treatment-independent hierarchical clustering histogram, all samples were

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grouped within their respective treatments and expression profiles of bulk/dissolved and control

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treatment were highly similar (Figure 1A). In the ENM treatment out of 570 and 1114 DEGs in

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shoots and roots, 365 and 485 were up-regulated and 205 and 629 down-regulated, respectively.

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In the bulk/dissolved treatment, out of 79 and 21 DEGS in shoots and roots, 57 and 7 were up-

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regulated and 22 and 14 down-regulated, respectively (Figure 1B). The Venn diagram shows 9 ACS Paragon Plus Environment


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that ENM and bulk/dissolved treatments shared only 53 DEGs for shoots and 7 for roots (Figure

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1B). To verify the reliability of the microarray results, gene expression levels were confirmed

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with qRT-PCR using 9 representative genes (4 for shoots, 5 for roots) (Figure 3). As depicted in

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Table S2, the trend in expression of all target genes showed consistency with result from the

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microarray analysis.

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The much greater number of DEGs and hence pathways and GOs affected in the ENM

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treatment is consistent with the results of our companion study where M. truncatula growth and

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nodulation were inhibited in response to the ENM treatment. In addition to differences in plant

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growth and nodulation, differences in microbial community composition were also observed

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between the ENM and bulk/dissolved treatments.18

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inhibition of nodulation and resultant effects on plant growth were due to toxicity to the plant or

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to the symbiont. Many of the responsive genes of M. truncatula in the ENM treatment are

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involved in nitrogen metabolism, nodulation, general stress responses and metal homeostasis

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indicating that the effects observed in the companion study could be explained by phytotoxicity.

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The fact that abundance of of S. meliloti in soil was not significantly different between ENM and

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bulk/dissolved treatments also supports this hypothesis.18

This raised the question of whether

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Several previous studies have been conducted on the phytotoxic effects of ENMs as assessed

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by whole-genome gene expression analysis. Landa et al. exposed Arabidopsis thaliana (A.

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thaliana) to ZnO NPs, fullerene and TiO2 NPs in a liquid growth media, and found that ZnO NPs

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had the strongest impact on gene expression profiles compared to the other two NPs, whereas

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TiO2 NPs elicited only weak transcriptional responses.30 Another study of A. thaliana grown on

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semisolid nutrient medium reported a high number of DEGs for both silver nanoparticles (Ag

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NPs) and Ag ions.31 However, these studies focused solely on pristine ENMs and corresponding


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metal ions, not the transformation products after wastewater treatment as in the present study.

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Ma et al. demonstrated that bulk Zn and Ag speciation were similar among control,

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bulk/dissolved and ENM biosolids used in the present study. They found that regardless of the

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form of metal introduced into the pilot WWTP, the metals were best modeled in XANES

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analysis as a mixture of ZnS, Zn3(PO4)2, Zn-FeOOH and Ag2S.11 After amendment to soil and

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aging outdoors in lysimeters for six months, the bulk speciation was still similar among the

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treatments where Zn was best modeled with a mixture of ZnS, Zn3(PO4)2, Zn-SiO2, and Zn-

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CH3CO2H.18 Despite these similarities in bulk chemical speciation, there was greater uptake of

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Zn and a greater reduction in nodulation in the ENM treatment than in the control or

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bulk/dissolved treatments.18 This suggests that some aspect of metal speciation not captured by

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XANES analysis differed among treatments, resulting in greater Zn bioavailability in the ENM

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treatment. In addition, the single chemical extraction method did not reveal any differences in

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metal bioavailability between ENM and bulk/dissolved treatments.

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The pathway most significantly influenced after KEGG analysis was that for oxidative

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phosphorylation, which was common to both ENM and bulk/dissolved treatments within

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exposed shoots (Table 2). The majority of genes involved in this pathway (31 and 8 DEGs in

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ENM and bulk/dissolved, respectively) were down-regulated in the shoots from both treatments,

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although the general changes in gene expression were rather weak (about 1.5~1.7 fold) in

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response to the bulk/dissolved exposure (File S3). This observation is consistent with a previous

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study showing a consistent down-regulation of this pathway as a result of AgNP, Ag bulk, and

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Ag+ exposure in zebrafish embryos.32 The two GOs associated with cellular nitrogen compound

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metabolic processes (GO:0034641) and electron transport (GO:0006118), were also enriched in

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response to both treatments. However, 44 and 62 genes were involved in these GOs in the ENM



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treatment, respectively, while only 5 genes were presented for both in the bulk/dissolved (File

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S2). Since adverse plant responses were only observed after exposure to ENM, we focus our

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discussion below on the distinct significant GOs, biological pathways, and individual genes

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induced in response to this treatment.

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metabolism, and stress are discussed.

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Nodulation

Genetic responses involved in nodulation, nitrogen

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GO analysis identified clusters of biological process and molecular function terms with many

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categories related to the observed effects of ENM exposure on growth and nodulation in M.

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truncatula.

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(GO:0009877), nodule morphogenesis (GO:0009878), and nitrogen fixation (GO:0009399) (File

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S2). In addition, Flavonoid metabolic/biosynthetic processes (GO:0009812, GO:0009813) and

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phenylpropanoid metabolic/biosynthetic processes (GO:0009698, GO:0009699) were among the

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significant metabolic processes affected by the ENM treatment; both of which are involved in

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stress responses33 and nodulation.34 Path Express also found that the flavonoid and isoflavonoid

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biosynthesis pathways were significantly altered (Table S3). Flavonoids play crucial roles in

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nodule development, particularly during signaling, infection thread development, and nodule

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organogenesis.35, 36 Some flavonoids are exuded by legume roots and act as signaling molecules

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which modify nodulation (nod) gene expression in rhizobia, thereby promoting nodule

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formation.37-39 Other flavonoids are involved in cellular changes in the root cortex and pericycle

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which are required for nodule organogenesis.40

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serving as ROS scavengers41 and are known to be involved in heavy metal stress responses in

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plants.42

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regulatory enzymes of flavonoid and isoflavonoid pathways in ENM exposed roots and some

Among GO categories identified in the ENM treatment are nodulation

Flavonoids also are antioxidant molecules

However, in our study, there was no increase in expression of genes encoding


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genes were differentially regulated in shoots, such as flavonoid 3'-hydroxylase (F3'H)

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(Mtr.13960.1.S1_at, 2.0 fold), and isoflavone reductase (IFR) (Mtr.410.1.S1_s_at, 1.9 fold). The

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up-regulation of these genes was confirmed independently with qRT-PCR (Figure 3A).

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Although gene enrichment analysis provides a quantitative method for discovering processes

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disrupted by xenobiotic exposure, this analysis is biased toward well-characterized genes and

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may ignore some other important DEGs that cause significant effects.

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Among these DEGs are nine highly (more than 100 fold) down-regulated genes that are

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involved in nodulation (Table 1), providing additional strong evidence that the lack of nodulation

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was largely due to plant responses to EMM exposure.

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(Mtr_3g055440),

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expression patterns and is known to be involved in the development, structure, maintenance and

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overall metabolism of the root nodule, had the greatest degree of down-regulation (-2547 fold).43,

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44

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their expression during root nodule development, were also highly down-regulated. For example,

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two members of the early nodulin gene family early nodulin (Mtr_1g030270) and early nodulin

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(ENOD)18 (MTR_7g065770) which are involved in initial signaling events, infection thread

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development, and nodule development,45 were both down-regulated by 265- and 334-fold,

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respectively. Importantly, the gene encoding the late nodulin leghemoglobin (Mtr_1g011540)

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was also down-regulated by 646-fold, and leghemoglobin is essential to nitrogen fixation in

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nodules due to its role in maintaining the micro-aerobic environment necessary to promote

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expression and activity of nitrogenase enzyme in rhizobia.46

The general nodulin gene

which encodes a nodule-specific plant protein with varying temporal

The other nodulin proteins, which are classified as “early” and “late” based on the timing of

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Apart from nodulin genes involved in the nodulation process, the nodulin 19 family plays an

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important role in plant stress responses47. The expression of one of the genes in this family (26



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fold, MTR_2g030460) was up-regulated by ENM exposure. Although a member of this stress

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protein family, the specific function of this gene is unknown. In addition, a cysteine cluster

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protein-coding gene (Mtr_1g042910) was strongly down-regulated by 1808 fold. This protein

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likely plays a role in M. truncatula response to stress conditions during symbiotic nitrogen

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fixation process.48

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Genes from two nodule-specific gene families which encode secreted peptides implicated in

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all stages of nodulation were also down-regulated in the ENM treatment.

These include

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Mtr_6g091470 (-314 fold) which encodes nodule-specific cysteine-rich peptide and two genes (-

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502 fold, Mtr_2g042470 and -226 fold, Mtr_2g042480) that encode nodule-specific glycine

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proteins 2D and 2A, respectively. A gene encoding a calmodulin-like protein (Mtr_3g055570)

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which is implicated in Ca2+-dependent signal transduction processes involved in root nodules

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functioning49 were strongly down-regulated by 589 fold in roots from the ENM treated plants.

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Recently, Syu et al reported that AgNPs antagonized ACC_Oxidase (1-aminocyclopropane-1-

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carboxylate oxidase), which inhibited root elongation in Arabidopsis seedlings, as well as

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reduced expression of ACC synthase 7 and ACC_Oxidase 2.50 Interestingly, both our microarray

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(58 fold) and qRT-PCR (64 fold) data (Figure 3) showed that a gene encoding an ACC_Oxidase -

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like protein associated with ethylene biosynthesis (MTR_2g069300) was up-regulated the

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strongest in the ENM exposed roots. Ethylene is a major plant hormone capable of influencing

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overall plant development and growth.51 Increased ethylene production is a known plant stress

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response to a number of heavy metals, including Zn.52 Additionally, the increased ethylene

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production is also known to restrict rhizobial infection and nodule formation.53

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Nitrogen Metabolism


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Nitrogen metabolism plays a key role in the overall plant physiology and health of

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legumes regardless of nodulation status. However, control of N cycling within the plant is

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obviously critical in regulating N fixation in legume-rhizobia symbiosis. There is evidence that

334

the ENM treatment influences many of the genes involved in that process. There are a total of

335

seven enzymes in the KEGG pathway of nitrogen metabolism that have been identified in M.

336

truncatula. Nine genes encoding five of these enzymes were significantly expressed (8 genes

337

down-regulated and 1 gene up-regulated) in roots from ENM exposed plants (Figure 2). Two

338

enzymes, ferredoxin-nitrite reductase and ferredoxin-dependent glutamate synthase, were not

339

affected by ENM exposure. Active nitrate transport and uptake by roots is the first step of

340

nitrogen acquisition in plants. The gene encoding the high-affinity nitrate transporter (Nrt) was

341

highly down-regulated by 21.5 fold, indicating a possible inhibition of extracellular nitrate and

342

nitrite uptake. Two members of a gene family associated with nitrate reductases (NR) were also

343

strongly down-regulated by 28.6 fold (MTR_3g073180) and 6.2 fold (MTR_5g059820)

344

respectively, providing another indication of possible reduced nitrate utilization. Both glutamine

345

synthetase (GS) (EC:6.3.1.2) and glutamate dehydrogenase (GDH) have been shown to be

346

involved in nitrogen assimilation in nodules.54 Then it is not surprising that we identified two

347

down-regulated (-1.7 fold and -1.8 fold) genes encoding the GS enzyme and one down-regulated

348

(-2.8 fold) gene encoding the GDH enzyme in unnodulated roots exposed to ENM. In addition,

349

we observed two genes that were down-regulated by 29.9 fold (MTR_3g077910) and 17.3 fold

350

(MTR_7g090950) and one gene that was up-regulated by 1.9 fold associated with carbonic

351

anhydrase, which has a possible role in nodule carbon dioxide metabolism possibly involved in

352

biochemical and physiological processes indirectly linked to nitrogen fixation and assimilation.55

353

These changes in nitrogen metabolism are likely related to the lack of nodulation observed in the



Page 16 of 32

354

ENM treatment. NO2 and NO3 concentrations were below detection limits in the soils and NH4

355

concentrations were similar among treatments.18

356 357

Stress Response and Metal Homeostasis Genes

358

The GO processes potentially related to toxicity and indicating stress responses, including

359

oxidative stress, in response to the ENM exposure included oxidoreductase activity

360

(GO:0016491), monooxygenase activity (GO:0004497), NADPH dehydrogenase (ubiquinone)

361

activity (GO:0008137), and NADPH dehydrogenase (quinone) activity (GO:0050136).

362

involvement of the transition metal ion binding (GO:0046914), transition metal ion transport

363

(GO:0000041), and metal ion binding (GO:0046872) processes provide strong evidence for the

364

role of metal ions in toxicity.

The

365

We observed differential expression of several genes involved in oxidative stress in the ENM

366

treatment. For example, one gene (Mtr_5g074710) encoding for a PEROX (peroxidase) was

367

highly up-regulated by 42 fold in the roots from ENM exposed plants. The expression was

368

confirmed by qRT-PCR (Figure 3B). This gene is known to play an important role in cellular

369

protection against oxidative stress associated with metal and metal NP toxicity by metabolizing

370

H2O2.31

371

In addition to the top DEGs listed above, 5 other up-regulated genes (about 2.3~10.0 fold,

372

from microarray result) and 10 down-regulated genes (about -1.5~ -9.0 fold) encoding

373

cytochrome P450 family proteins were identified in the roots (File S1), some of which may be

374

related to general stress. Expression of one of the P450 family (P450 71B10, Mtr.12616.1.S1_at)

375

as well as GST (Mtr.43621.1.S1_at), which are also implicated in stress responses (e.g., oxidative

376

damage, pathogens, herbicides),41 was confirmed with qRT-PCR (Figure 3A). Interestingly, one


Page 17 of 32


377

of the up-regulated (27 fold, Mtr_7g038480) genes is associated with NADPH oxidase, which

378

generates superoxide radicals (O2.-) that can be converted to H2O2. The up-regulation of this

379

gene was confirmed with qRT-PCR (Figure 3B). Induction of NADPH oxidase yields increased

380

ROS production, which is thought to play a role in immune response toward pathogens in

381

Arabidopsis.56

382

The differential expression of several genes involved in metal homeostasis, in particular Zn

383

homeostasis, were also found, suggesting that Zn ions played an important role in the observed

384

toxicity. Several metal homeostasis genes encoding proteins were also highly up-regulated in the

385

roots from plants grown in the ENM treatment (Table 1), including cation diffusion facilitator57

386

(29 fold, Mtr_3g080090), metal tolerance protein (MTP)58 (14 fold, Mtr_5g075680), and metal

387

transporter (MTR)59 (13 fold, Mtr_3g088460). These genes have been shown to be involved in

388

metal binding, transport, or storage. It has been suggested that MTP and MTR have a role in

389

general Zn homeostasis and tolerance to Zn excess in M. truncatula.58, 59 The expression levels

390

of these two genes were also independently confirmed with qRT-PCR in this study (Figure 3B).

391

Arrivault et al. 60 observed that an ectopic over-expression of an MTP homologue was associated

392

with Zn accumulation in both roots and leaves of Arabidopsis exposed to ZnSO4.

393

observations are consistent with our findings, as we observed strong up-regulation in response to

394

ENM exposure. Given that the concentration of Zn uptake in shoots from ENM treatment are

395

significantly higher than in the bulk/dissolved treatment, which indicates the accumulation of Zn

396

ions derived from nanoparticles may trigger the expression of metal homeostasis genes in M.

397

truncatula.

These

398

Photosynthesis was also identified as an affected pathway in shoots exposed to ENM treatment,

399

and its efficiency may be drastically reduced due to abiotic stress.61 Twenty DEGs involved in



Page 18 of 32

400

photosynthesis pathways were down-regulated in the shoots from plants exposed to ENM. The

401

observed slight down-regulation of genes involved in photosynthesis is consistent with Simon et

402

al, who reported expression of photosynthesis-related genes was slightly decreased in

403

Chlamydomonas reinhardtii exposed to ZnO NPs or AgNPs at 1 mg L-1 and drastically

404

decreased during exposure to 1 mg L-1 of TiO2 NPs.62

405

In summary, this study provides the first comprehensive insight into the toxicogenomic

406

responses of M. truncatula grown in soils amended with aged biosolids containing a mixture of

407

ENMs (Ag, TiO2 and ZnO). Considering the results from the companion study,18 the gene

408

expression patterns are consistent with the hypothesis that inhibition of nodulation by ENM

409

exposure was a result of plant toxicity rather than microbial toxicity, particularly since

410

population densities of S. meliloti were similar in the bulk/dissolved and ENM treatments. We

411

identified multiple genes involved in nodulation and inorganic nitrogen metabolism that were

412

down regulated. In addition, genes involved in oxidative stress response were up-regulated. The

413

companion study showed that Zn concentrations and uptake were higher in shoots from the ENM

414

treatment than in the bulk/dissolved treatment while Ti and Ag concentrations were not

415

significantly different.18 The present study showed that several genes involved in metal binding

416

and Zn homeostasis were up-regulated. Taken together, these findings suggest that inhibition of

417

growth and nodulation in M. truncatula exposed to ENM treatment is likely the result of

418

enhanced bioavailability of Zn ions in the biosolids-amended soil containing aged ENMs

419

resulting in phytotoxicity.

420 421

ACKNOWLEDGMENTS

422

The authors acknowledge the advice and assistance of D. Wall and K-C Chen (Microarray Core Facility)

423

and J.V. Kupper (Rhizosphere Science Laboratory). This research was funded by a grant from the U.S. 18 ACS Paragon Plus Environment

Page 19 of 32


424

Environmental Protection Agency's Science to Achieve Results 63 program (RD834574). J. Unrine and O.

425

Tsyusko were also supported by the National Science Foundation (NSF) and the Environmental

426

Protection Agency (EPA) under NSF Cooperative Agreement EF-0830093, Center for the Environmental

427

Implications of NanoTechnology (CEINT). Any opinions, findings, conclusions or recommendations

428

expressed in this material are those of the author(s) and do not necessarily reflect the views of the NSF or

429

the EPA. This work has not been subjected to NSF or EPA review and no official endorsement should be

430

inferred.

431 432

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Page 26 of 32

Figure 1. Transcriptome analysis of Medicago truncatula tissues in response to exposure to control, bulk/dissolved and ENM (engineered nanomaterial) treatments. A) Hierarchical cluster analysis of differentially expressed genes (DEGs) (≥1.5-fold up/downregulated, p < 0.05) in (a) shoots and (b) roots from M. truncatula exposed to control, bulk/dissolved and ENM treatments. Color bars indicate the gene expression levels, where red represents up-regulated genes and blue represents down-regulated genes. Numbers above the columns refer to the individual replicate number. B) Venn diagram showing numbers of unique DEGs and common DEGs derived from M. truncatual (a: shoots, b: roots) after exposure to ENM and bulk/dissolved treatments as compared to control. DEGs were included when P < 0.05 and fold change of up/down-regulation > 1.5. Indicated in the diagram are the number of up-regulated and down-regulated DEGs; C) The number and fold change distribution of DEGs in tissues of M. truncatula (a: shoots, b: roots) exposed to ENM versus control treatment. 26 ACS Paragon Plus Environment

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Figure 2. Nitrogen metabolism of Medicago truncatula with associated differentially expressed genes in the roots from plants exposed to ENM (engineered nanomaterial) treatment. A) List of differentially expressed genes (DEGs) associated with nitrogen metabolism pathway as identified by ENM exposure. B) Nitrogen metabolism pathway leading to metabolic enzyme encoding genes found differentially expressed in the roots from M. truncatula exposed to ENM. (Adapted from the metabolic pathway maps in KEGG pathway database.27, 28 http://www.genome.jp/kegg-bin/show_pathway?mtr00910) 27 ACS Paragon Plus Environment


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10

Fold Change

8

A. Shoots

Control Bulk/Dissolved ENM

*

6

*

4

*

*

*

2

*

0 IFR

F3'H

GST

P450

Genes 80 B. Roots

Control Bulk/Dissolved ENM

*

Fold Change

70

60 30 * 20

10

*

*

*

0 MTP

MTR

PEROX

NADPH

ACC_Oxidase

Genes

Figure 3. Independent qRT-PCR confirmation of expression levels for representative genes in shoots and roots from Medicago Truncatula exposed to control, bulk/dissolved and ENM treatment (A: four genes in shoots, B: five genes in roots). Gene expression were normalized using Actin 2 mRNA. Data are presented in arbitrary unit compared to control. qPCR data represent the mean ± SEM (standard error of the mean) of n = 5 individual shoots (n = 3 individual pooled roots) , * p < 0.05. Abbreviations include IFR, isoflavone reductase; F3’H, 28 ACS Paragon Plus Environment

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flavonoid 3'-hydroxylase; GST, glutathione S-transferase; P450, cytochrome P450 71B10; MTP, metal tolerance proteins; MTR metal transporter; PEROX, peroxidase; NADPH, nicotinamide adenine dinucleotide phosphate-oxidase; ACC_Oxidase, 1-aminocyclopropane-1-carboxylate oxidase-like protein. The horizontal line indicates control expression (1-fold).



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Table 1. Top 10 unique genes significantly up- or down-regulated in roots following ENM (engineered nanomaterial) treatment. Gene symbol Up-regulated: MTR_2g069300 MTR_5g074710 MTR_3g080090 MTR_7g038480 MTR_2g030460 MTR_7g100080 MTR_1g092870 MTR_5g075680 MTR_8g018110 MTR_3g088460 Down-regulated: MTR_3g055440 MTR_1g042910 MTR_127s0023 Mtr_1g011540 MTR_3g055570 MTR_2g042470 MTR_7g065770 MTR_6g091470 MTR_1g030270 MTR_2g042480

ENM Fold-change P-value

Gene description 1-aminocyclopropane-1-carboxylate oxidase-like protein Peroxidase Cation diffusion facilitator NADPH oxidase MtN19 protein Zinc finger protein Hippocampus abundant transcript-like protein Metal tolerance protein F-box protein Metal transporter Nodulin Cysteine cluster proteins (CCP) Limonene synthase Leghemoglobin Calmodulin-like protein Nodule-specific glycine-rich protein 2D Early nodulin (ENOD)18 Nodule-specific cysteine-rich peptide Early nodulin Nodule-specific glycine-rich protein 2A

Bulk/Dissolved Fold-change

57.9 42.2 29.4 27.3 26.3 17.9 15.4 14.3 13.1 12.7

4.9E-06 1.8E-05 1.1E-04 4.0E-04 9.9E-05 7.9E-05 2.3E-04 8.7E-05 7.4E-05 5.4E-05

-1.41 -1.53 -1.18 -1.55 -1.49 1.55 -1.18 -1.15 1.06 -1.25

-2546.8 -1807.8 -1023.6 -646.3 -588.6 -501.9 -333.7 -313.9 -264.5 -226.4

9.9E-07 4.9E-06 1.9E-06 7.6E-05 7.8E-05 2.4E-05 7.4E-05 2.0E-05 3.9E-06 2.7E-04

-1.17 -1.19 -1.11 -1.08 -1.18 -1.21 -1.22 -1.05 -1.10 -1.00

Note: the top 10 unique genes were significant (P < 0.05) in roots exposure to ENM compared to control treatment, whereas no significant difference in bulk/dissolved treatment were observed.


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Table 2. Significant pathways after KEGG analysis of DEGs (P ≤ 0.05 and ≥ ±1.5 fold change) in the shoots and roots from Medicago truncatula exposed to ENM (engineered nanomaterial) and bulk/dissolved treatment compared to the control.

Genes in the pathway

Pathway name

Number of DEGs Down Up

Enrichment score

Enrichment p-value

ENM (shoots): Oxidative phosphorylation Photosynthesis Metabolic pathways Tryptophan metabolism Linoleic acid metabolism Flavonoid biosynthesis

31 20 94 4 5 6

31 20 63 1 4 0

0 0 31 3 1 6

37.1 22.6 16.6 4.9 4.6 4.3

7.5E-17 1.6E-10 6.1E-08 7.0E-03 1.0E-02 1.0E-02

Bulk/Dissolved (shoots): Oxidative phosphorylation Ribosome

8 5

8 5

0 0

14.0 3.8

8.2E-07 2.0E-02

22 9 7 14 115 11 7 20 3 12 3 3

8 8 0 3 46 1 3 3 2 4 2 3

14 1 7 11 69 10 4 17 1 8 1 0

19.7 14.0 13.7 13.6 9.3 7.4 4.7 4.5 4.1 4.0 3.5 3.1

2.7E-09 8.4E-07 1.1E-06 1.2E-06 9.3E-05 6.0E-04 9.0E-03 1.1E-02 1.6E-02 1.9E-02 3.0 E-02 4.7E-02

ENM (roots): Photosynthesis Nitrogen metabolism Photosynthesis - antenna proteins Glyoxylate and dicarboxylate metabolism Metabolic pathways Carbon fixation in photosynthetic organisms Alanine, aspartate and glutamate metabolism Carbon metabolism Tropane, piperidine and pyridine alkaloid biosynthesis Phenylalanine metabolism Isoquinoline alkaloid biosynthesis Vitamin B6 metabolism



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TOC ART


Toxicogenomic Responses of the Model Legume Medicago truncatula

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