Effect of Gallium Exposure in Arabidopsis thaliana is Similar to

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Effect of gallium exposure in Arabidopsis thaliana is similar to aluminum stress Hsin-Fang Chang, Shan-Li Wang, and Kuo-Chen Yeh Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b05760 • Publication Date (Web): 15 Jan 2017 Downloaded from http://pubs.acs.org on January 15, 2017

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Environmental Science & Technology

1

Title: Effect of gallium exposure in Arabidopsis thaliana is similar to aluminum

2

stress

3

Hsin-Fang Chang1,2, Shan-Li Wang1 and Kuo-Chen Yeh2*

4

1

5

TAIWAN

6

2

7

TAIWAN

Department of Agricultural Chemistry, National Taiwan University, Taipei 106,

Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115,

8 9 10 11 12

* Corresponding author:

13

Kuo-Chen Yeh

14

Agricultural Biotechnology Research Center, Academia Sinica 128

15

Academia Road Section 2, Taipei, Taiwan 11529

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Tel: 886-2-2787-2056

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Fax: 886-2-2651-5600

18

Email: [email protected]

19

ACS Paragon Plus Environment


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Abstract: Although gallium (Ga) is a rare element, it is widely used in semiconductor

21

devices.

22

semiconductor-producing countries. Here, the physiological and molecular impacts of

23

Ga in the model plant Arabidopsis thaliana were investigated in medium culture. The

24

primary symptom of Ga toxicity is inhibition of root growth. The increased production

25

of malondialdehyde (MDA) suggests that Ga stress could cause oxidative damage in

26

plants. Roots were the main Ga accumulating sites. The distinctive Ga granules were

27

deposited within the intercellular space in roots. The granules indicate Ga(OH)3

28

precipitation, which indicates immobilization or limited translocation of Ga in A.

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thaliana. Ga stress induces root secretion of organic acids such as citrate and malate.

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The expression of the transporters AtALMT and AtMATE, responsible for citrate and

31

malate secretion, respectively, were elevated under Ga stress, so the secretion may

32

play a role in the resistance. Indeed, supplying exogenous citrate significantly

33

enhanced Ga tolerance. The overall response to Ga exposure in A. thaliana is highly

34

similar to that with aluminum stress. Our findings provide information for risk

35

assessment in Ga-contaminated soil.

Ga

contamination

of

the

environment


has

been

found

in

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Introduction

37

Gallium (Ga) is one of the major elements in semiconductor compounds that are

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used in integrated circuits and optoelectronic devices.1 In satisfying the demands for

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semiconductor compounds, the world Ga production was estimated to be 435 t in

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2015.2 Taiwan is the largest producer of wafer foundry and integrated circuit ackaging

41

and testing, accounting for 76% and 56% of total worldwide production,

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respectively.3 The low processing efficiency of Ga has resulted in inevitable discharge

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of Ga into the environment.4 Chen5 indicated that the concentration of Ga in

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groundwater samples collected near the Science-based Industrial Park in Taiwan was

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7.91 to 41.49 µg L-1 as compared with background values of < 1 µg L-1. Although the

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environmental fate of Ga is not well understood, the Environmental Protection

47

Administration of Taiwan has taken precautionary steps to regulate the maximum

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contamination level of Ga (i.e., 0.1 mg L-1) in effluent wastewater from

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optoelectronics manufacturers and science parks.6 Because Ga is not an essential

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element for living organisms, concerns about the environmental risks and human

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health threat with Ga contamination have been frequently raised.7-9

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The Ga contamination in soil and water may lead to its uptake by plants and

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accumulation of Ga in the food chain, however the (eco)toxicological effects of Ga

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have been little investigated. Previous studies reveal that excessive intake of Ga by

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animals resulted in the accumulation of Ga in bones and led to impaired kidney

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function and failure of the nervous system.10, 11 Ga appears to inhibit DNA synthesis

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by its action on ribonucleotide reductase12 and it interferes with the cellular immune

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function.13 Ga has antimicrobial effects to inhibit the growth of pathogenic bacteria

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(Pseudomonas aeruginosa) and prevents biofilm formation, so Ga is a promising new

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therapeutic agent against bacterial infections.14



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Kopittke and Yermiyahu (2007) found that Ga had negative effects on root

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elongation and morphology in cowpea.15 Relative growth rate, transpiration rate and

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water-use efficiency decreased with increasing Ga concentration in rice seedlings

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(Oryza sativa L. cv. XZX45).16 Moreover, overaccumulation of Ga in rice (O.

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sativa L. cv. XZX45) provoked the formation of DNA-protein cross-links in roots,

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which resulted in cell death and growth inhibition of rice seedlings.17 In contrast, a

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previous study suggested that Ga may have a beneficial role in the growth of

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microorganisms and some fungi, such as Aspergillus niger.18 Ga concentration less

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than 10 mg L-1 stimulated the growth of rice seedlings (O. sativa L. cv. Taikeng 9).19

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As well, Ga2(SO4)3 inhibited the Fe deficiency stress response in cucumber.20

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However, information on Ga uptake and accumulation in plants is limited. To further

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understand the toxic effects and possible resistance response with Ga exposure, the

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underlying physiological and molecular mechanisms must be investigated.

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Plants have evolved a complex network of metal transport, chelation, and

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intracellular sequestration processes to ameliorate the harmful effects of metals. The

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detoxification strategies at the cellular level include the utilization of enzymatic and

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non-enzymatic components in an antioxidative defense system.21 The strategies are

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involved in a complex cooperative network to reduce the metal-induced damage and

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pro-oxidant

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mechanisms.23 In particular, glutathione (GSH) and phytochelatins (PCs) play a

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pivotal role in defense against heavy metal-induced oxidative damage. GSH

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(γ-glutamylcysteintlglycine) is a key metal scavenger because its thiol (-SH) group

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has high affinity to metal ions. It also acts as a primary precursor for PC synthesis.

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PCs could form complexes with toxic metal ions in the cytosol for subsequent

85

transport into the vacuole.24 In addition, accumulation of metals by metal

conditions

in

plants.22

Plants

share

common


metal-tolerance

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compartmentalization in apoplast tissues such as trichome and cell walls could

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prevent the transportation of metals across the plasma membrane.25 Excess metal ions

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are removed from the cytosol by efflux (metal exclusion).

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In this study, we used Arabidopsis thaliana (A. thaliana) as the model plant to

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investigate the effect of Ga exposure on plants. A. thaliana is considered an excellent

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model plant for analysis of biological processes in plants because its life cycle is

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relatively short and its genomic sequencing has been completed. We measured

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representative characteristics such as biomass, root length, lipid peroxidation and

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element content to evaluate the toxicity response to Ga stress. Transmission electron

95

microscopy with energy-dispersive X-ray spectroscopy (TEM-EDX) and X-ray

96

absorption (XAS) were used to determine the spatial distribution and chemical

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speciation of Ga within plant tissues and to provide direct information for clarifying

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the detoxification mechanisms of Ga in plants. Furthermore, we investigated the role

99

of secreted organic acids in regulating tolerance of Ga. The information we provide

100

may be helpful in clarifying the toxicity and resistance response of plants to Ga at

101

physiological and molecular levels for lowering the risk from Ga environmental

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contamination.

103



104 105

Materials and Methods Plant materials and stress conditions. Arabidopsis thaliana (ecotype Columbia)

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seeds were surface-sterilized with 70% ethanol for 2 min, treated with 1.2% bleach

107

containing 0.02% SDS for 15 min, then rinsed with sterilized Millipore water 6 times

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and placed at 4oC in the dark for 3 d for stratification. The sterilized seedlings were

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grown on half-strength (1/2) MS medium (Murashige & Skoog, 1962) containing 1%

110

sucrose, 0.5 g/L 2-morpholinoethanesulfonic acid, and 0.3% phytagel at pH 5.7 in

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Petri dishes for 7 d. Plantlets were then transferred to fresh medium containing

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Ga(NO3)3 and grown vertically in 1/2 MS medium with 0.7% agar at 22oC with a

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16-h light (intensity = 70 µmol m-2 sec-1) and 8-h dark cycle for 8 d. The

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concentration of Ga in the medium ranged from 6 to 750 µM. Quadruplicate sets of

115

10 plants per treatment were used. The fresh weight of the plants in each replicate was

116

measured. Root length measurements were analyzed by using ImageJ 1.37v

117

(http://rsb.info.nih.gov/ij/).

118

Lipid peroxidation estimation. Malondialdehyde (MDA), indicative of lipid

119

peroxidation, was measured by using the thiobarbituric acid reactive substances

120

(TBARS) assay, according to Heath and Packer (1968),26 with slight modification.

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Plants (80-100 mg) were homogenized in liquid nitrogen, then 1 mL of 0.1% (w/v)

122

trichloroacetic acid (TCA) was added. The extracts were centrifuged at 15,700 ×g for

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10 min at 4°C, then 400 µL supernatant was pipetted into a solution containing 1 mL

124

of 0.5% (w/v) 2-thio-barbituric acid and 1 mL of 20% (w/v) TCA. The mixture was

125

heated to 80°C for 30 min and the reaction was arrested by quickly transferring the

126

mixture to an ice bath. The absorbance of the supernatant was read at 532 and 600 nm

127

by using a UV−Vis spectrophotometer (BioTek MultiFlo, Winooski, VT).


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Determination of Ga concentration in plant samples. For sample preparation,

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shoot and root tissues were washed in 3 successive baths of water, 10 mM CaCl2, and

130

water, then dried at 70oC for 3 d. The samples of 0.1 g were weighed and transferred to

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Teflon vessels, and 2.0 mL of 65% HNO3 and 0.5 mL H2O2 were added to each vessel.

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The samples were digested at 195oC for 15 min by using the MarsXpress microwave

133

digestion system (CEM, Matthews, NC, USA). Then samples were cooled to room

134

temperature and filtered by using a 0.45-µm-membrane filter. The concentration of

135

elements was analyzed by inductively coupled plasma-optical emission spectrometry

136

(ICP-AES; Perkin Elmer OPTIMA 5300). The wavelength at 417.206 nm was selected

137

for Ga measurement, with limit of detection (LOD) 0.03 mg/L. A multi-element stock

138

standard solution (ICP Multi Element Standard Certipur VIII; Merck) was used for

139

calibration curves and quality control (QC) in ICP measurement. For the quality of the

140

elemental analysis was verified by using Fe in NIST SRM 1573a because of no

141

certified reference material for Ga. The recovery rate of Fe was determined to be

142

92%.

143

Scanning transmission electron microscopy and energy-dispersive X-ray

144

spectroscopy (STEM-EDX). Ga distribution at the cellular scale was investigated by

145

using a field emission gun STEM operating at 200 kV (FEI Tecnai G2 20 S-Twin) and

146

equipped with an energy dispersive x-ray analyzer (EDX, METEK). Root

147

cross-sections were prepared by high-pressure freezing,27 post-fixed with osmium

148

tetroxide and infiltrated in Spurr’s embedding medium. Ultrathin sections (100 nm)

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were prepared by use of an ultra-microtome (LEICA, EMKMR2) and stained with

150

uranyl acetate for observation by STEM.

151

Ga K-edge XAS analysis. The plants grown in agar containing 500 µM Ga were

152

selected for Ga K-edge XAS analysis. The samples were washed and lyophilized in a



153

Labconco freeze-dryer at -80◦C, then ground to a fine powder with use of a mortar and

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pestle and compressed into 9-mm pellets under 5 tons. Ga K-edge XAS analysis was

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conducted at Beamline X17C in the National Synchrotron Radiation Research Center

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(NSRRC), Hsinchu, Taiwan. The spectra were collected in fluorescence mode using a

157

Lytle detector with a 6-µm zinc filter and a set of Soller slits. At least three scans

158

were collected and averaged for each sample, and the spectrum of a metallic Ga film

159

was simultaneously collected in each scan for energy calibration to 10367 eV (i.e., E0).

160

After baseline correction and normalization, the k3-weighted EXAFS spectra in the

161

region of k = 1-12 Å−1 were extracted for linear combination fitting (LCF) by using

162

Athena software28 and a set of reference spectra. The reference compounds used in LCF

163

were Ga(NO)3(s), Ga(NO)3(aq), Ga-citrate(aq), Ga-PO4(aq), Ga(OH)3 and Ga-cellulose. The

164

LCF procedure was performed for each EXAFS spectrum using all possible binary

165

combinations of the reference compounds. The goodness of fit was evaluated by the R

166

factor, defined as Σ(data-fit)2/ (data)2 (see Athena Users’ Manual for details), with

167

sums over the data points in the fitting region. To avoid overfitting with a large number

168

of reference compounds, adding a third reference compound to LCF was only allowed

169

when the added compound accounted for over 10% of Ga in the samples and the R

170

value of the fitting decreased.

171

RNA isolation and quantitative real-time RT-PCR. For real-time quantitative

172

RT-PCR (qRT-PCR), Seven-day-old A. thaliana seedlings were transferred to 0

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(control) and 250 or 500 µM Ga-treated medium with and without exogenously

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supplied citrate for 1, 3, 5 and 7 d. RNA extraction and reverse transcription were

175

performed as described.29 Sequence data from this study were deposited at

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GenBank/EMBL [accession nos. At1g51340 (MATE), At1g08430 (ALMT1) and

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At5g25760 (UBC21)].


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Collection of root exudates and metabolite-profiling analysis. Samples of

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21-day-old A. thaliana were transferred to 1/2 Hoagland’s nutrient solution (pH4.2)

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with 500 µM Ga(NO3)3 or AlCl3 for 2 d. Briefly, for exudate collection, the plants

181

were removed from nutrient solution, and plant root systems were submerged in 5 mL

182

distilled deionized water for 4 h. The organic acids concentrations in root exudates

183

were determined by Waters Acquity UPLC (Waters, Parsippany, NJ, USA) with a

184

BEH 1.7-µm RP-18 column (1.7 µm, 2.1 x 100 mm; Waters) interfaced to a TSQ

185

Quantum Access Max MS (ThermoFisher Scientific, Waltham, MA, USA). The

186

LC-MS/MS conditions and the sample derivatization method for analysis were as

187

described.30 MS data were processed by using MarkerLynx XS 4.1 SCN639 within

188

MassLynx 4.1 (Waters).

189

Statistical analysis. Data are separately reported for shoot and root tissue and

190

were analyzed in quadruplicate for the assays described above. One-way ANOVA

191

followed by Student-Newman-Keuls multiple comparison test (p < 0.05) was used to

192

determine differences among treatments.

193 194

Results and Discussion

195

Phenotype characterization of Ga-treated A. thaliana. To determine the Ga

196

concentrations that cause toxic effects in A. thaliana, we tested growth with Ga(NO3)3

197

at 0 to 750 µM (Figure 1). The root growth was slightly repressed with 6, 30 and 150

198

µM Ga(NO3)3 (Figure 1B) and was inhibited with an increase in Ga concentration.

199

With 250 and 500 µM Ga(NO3)3, the mean root length was reduced by nearly 30%

200

and 60%, respectively. Consistent with the root phenotype, 250 and 500 µM

201

Ga(NO3)3 significantly reduced the fresh weight of shoots. However, low

202

concentrations (6, 30 and 150 µM) of Ga(NO3)3 conferred no significant difference in



203

shoot biomass (p < 0.05; Figure 1D). At 750 µM Ga(NO3)3, A. thaliana root growth

204

was stunted and failed to penetrate the medium. Ga toxicity was found more in the root

205

length than shoot weight (Fig. 1), a phenotype that is similar to Al toxicity to plants.31, 32

206

The calculated EC10 for the inhibition of growth and root elongation was 36.6 and

207

3.66 µM, respectively. The Ga concentration was higher in roots than shoots (Table

208

S1). For example, up to 935.35 and 173.15 mg g-1 DW of Ga accumulated in roots

209

and shoots, respectively, with medium containing 750 µM Ga. However, Ga

210

concentration was below the detection limit with 30 µM Ga treatment (Figure S1).

211

Effect of Ga on intracellular MDA levels. The cytotoxicity of Ga was further

212

verified by measuring MDA content, generally considered an important indicator of

213

intracellular oxidative stress in plants. At 250 to 500 µM Ga(NO3)3, the MDA

214

production in roots was increased by 31% and 47%, respectively, relative to the

215

control plants (Figure S2). On the other hand, the exposure to Ga(NO3)3 had no effect

216

on MDA content in shoots.

217

After exposure to a Ga concentration of 250 µM or higher, the MDA content in

218

roots significantly increased (Figure S2). The elevated MDA level indicated that Ga

219

induced oxidative stress in roots, which could lead to cell damage. Such abiotic stress

220

damage is consistent with the phenotype observations and biomass results (Figure 1),

221

suggesting that the Ga-mediated oxidative stress could be the mechanism underlying

222

the etiology of Ga-associated toxicity. Comparatively, the MDA formation in shoots

223

was insignificant, so the oxidative stress was not induced because Ga did not reach the

224

shoot or was detoxified in shoot tissues. As revealed in results of Ga concentration in

225

plant tissues (Figure S1 and Table S1), most of the Ga (60-80%) was sequestered in

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roots, and less (20-40%) Ga was transported to shoots. Plants growing on toxic

227

metalliferous environments cannot prevent metal uptake but only immobilize it and,


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228

hence, accumulate metals in their tissues to varying degrees.33 The earliest Ga-toxicity

229

response is the inhibition of root growth, which is associated with gross changes in

230

root morphology and results in inhibited root elongation.

231

Toxic metals (i.e., Cd) are known to induce stress effects in plants.34 Metal(s) that

232

are redox-active (i.e., Cu) could produce reactive oxygen species (ROS) directly and

233

cause immediate anti-oxidative responses as well as damage to cellular components

234

and structures.35 However, Ga is not redox-active and could result in oxidative stress

235

via indirect mechanisms.36

236

Cellular antioxidant defense under Ga stress: role of glutathione (GSH) and

237

phytochelatin (PCs). To explore the mechanistic connection between GSH/PC levels

238

and Ga stress tolerance in A. thaliana, we examined cad1-3 and cad2-1 mutants,

239

which had been genetically modified to alter GSH/PC levels. The cad1 locus is

240

deficient in PC biosynthesis with a mutation in PC synthase gene,37 and the cad2–1

241

has 40% of wild-type levels of GSH and is therefore PC-deficient.38, 39 These mutants

242

are sensitive to a range of heavy metals. Thus, the physiological responses of cad1-3

243

and cad2-1 under Ga treatment may be helpful in clarifying whether A. thaliana use

244

GSH/PCs to detoxify the Ga stress. These mutants were not more hypersensitive to

245

Ga stress than the wild type (Figure S3). Therefore, GSH/PCs play an insignificant

246

role in the tolerance of plants to Ga stress.

247

Subcellular localization and speciation of Ga in A. thaliana roots. To further

248

investigate whether apoplastic regulation plays an important role in Ga toxicity,

249

STEM was used to obtain a subcellular image of Ga micro-distribution in A. thaliana

250

roots. We observed distinctive granular deposits (bright spots) within the intercellular

251

space and/or binding to cell walls of the cortical tissue in plants exposed to 500 µM

252

Ga (Figure 2A-B). EDX spectra of these granular deposits showed the presence of Ga:



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253

Ga Kα1(9.252) and Ga Kβ1(1.098). Furthermore, in plants exposed to a high

254

concentration of Ga (500 µM), the intercellular space of the cortex was the only site

255

with a detectable amount of Ga (Figure 2A-B, (a) and (d)). Intercellular

256

compartmentalization could be considered an extracellular barrier, exploited for

257

protecting against excess metal exposure in the environment. Therefore, by increased

258

binding of Ga to the cell wall, reduced bioactivity via precipitation in the intercellular

259

space, or excluding the metal out of the cell with efflux pumps, intercellular

260

compartmentalization would be an efficient strategy to prevent metals from entering

261

into cells or keeping a low concentration of toxic metal ions in the cytoplasm.25 In giant

262

algal cells of Chara coralline (C. coralline), up to 99% of total Ga is in cell walls,

263

which suggests that the cell wall is an important site for binding Ga because of its

264

high density of negative charges.40

265

The Ga K-edge EXAFS spectra of various Ga standards are in Figure S4. The

266

spectra of Ga(III) were sensitive on the bonding configuration; however, the standard

267

complexes with the same functional groups in different ligands shown similar EXAFS

268

spectra. For example, the spectra of Ga-cysteine(aq), Ga-glycine(aq) and Ga-histidine(aq)

269

(Ga-S/O/N) were highly similar to those of Ga-citrate(aq) and Ga-malate(aq) (Ga-simple

270

organic

271

Ga-carboxyl/hydroxyl structure, referred as “Ga-COOH/OH” in Figure S4.

272

Meanwhile, in considering the possibility of Ga absorption on the cell wall or

273

sequestration in apoplasts, the spectra of Ga-cellulose(s) or Ga(OH)3(am) and Ga(NO)3(s)

274

were selected as references for the Ga(III) absorbed form or hydroxide precipitates,

275

respectively. Therefore, the Ga standards for the fingerprint approach for EXAFS

276

analysis were Ga(NO)3(s), Ga(NO)3(aq), Ga-citrate(aq), Ga-PO4(aq), Ga(OH)3 and

277

Ga-cellulose.

acid).

Thus,

only

Ga-citrate

was

selected


to

represent

the

Page 13 of 29


278

Figure 3 shows the k3-weighted Ga EXAFS spectra in A. thaliana roots and

279

shoots. For plants with 500 µM Ga treatment, the best two-component fits with the

280

mixture of Ga(OH)3(s) and Ga-citrate. Both the spectra were found to be similar,

281

indicating that Ga speciation was not altered during translocation. Ga(OH)3 represents

282

52.2% total Ga in roots (Table 1). The pH of the root cell wall of A. thaliana is

283

maintained at approximately 5.3 to 5.5.41 Ga tends to form Ga(OH)3 in solution and

284

with an expected upper limit of solubility of 10-7.2 M at pH 5.2.7 According to the LCF

285

result (Table 1 and Figure 3), we could conclude that the Ga-containing granular

286

deposits observed in the STEM image was determined to be Ga(OH)3 precipitates

287

(zoom and spectra in Figure 2A). Hence, the Ga-rich granular structures in the

288

intercellular space (Figure 2A-B) is the main storage compartment for excess Ga

289

accumulation and precipitate in the intercellular space could be one of the main

290

mechanisms in the detoxification of Ga. The second Ga species in the roots and

291

shoots was Ga-citrate, representing Ga(III) bound to COOH/OH ligands. This

292

indicated that Ga(III) is in the form of complexes with small organic ligands, most

293

likely citrate or malate. Meanwhile, minor amounts of Ga(III) bound to S-containing

294

ligand was detected. This finding was consistent with the observation that GSH/PCs

295

(as a complex with S ligands) did not play a role in defense of Ga-induced stress as

296

mentioned earlier. However, further studies are needed to confirm the possibility of

297

internal detoxification of Ga, which is relatively non-toxic comparing to the free Ga

298

ion despite its only loose association with organic acids.

299

Mechanism of Ga detoxification: the possible role of organic acids. Ga and Al

300

both belong to Group 13 in the periodic table. Therefore, Ga3+ has similar toxic

301

effects as Al3+ on yeast cells. In considering the similar chemical properties of Ga3+

302

and Al3+, we hypothesized that the detoxification mechanism of Al3+ in A. thaliana



303

may also play a role of detoxifying Ga3+ in A. thaliana. Currently, Al-activated

304

exudation of organic acid (OA) anions from root apices is the best-documented and

305

-characterized plant Al-tolerance mechanism. In the rhizosphere, the released OAs

306

from roots such as citrate, malate and oxalate can interact with the Al ion to form

307

non-toxic complexes.40 Therefore, we investigated the effect of Ga stress on Al

308

tolerance-related gene expression and citrate.

309

Al-activated root malate and citrate exudation play an important role in plant Al

310

tolerance. The previously characterized MULTIDRUG AND TOXIC COMPOUND

311

EXTRUSION (MATE), a homolog of the recently discovered sorghum and barley

312

Al-tolerance genes,42, 43 encodes an Al-activated citrate transporter in A. thaliana.

313

MATE, together with AL-ACTIVATED MALATE TRANSPORTER1 (ALMT1),

314

encoding an Al-activated A. thaliana malate transporter, can protect A. thaliana roots

315

against Al toxicity by facilitating root citrate and malate exudation, respectively.44, 45

316

The results showed that Ga stress rapidly induced the expression of AtALMT1 and

317

AtMATE in A. thaliana roots (Figure S5).

318

The profile of organic acid release during the 2 d period showed that citrate was

319

the predominant organic acid released by Ga-treated roots, along with a slight

320

increase in malate, pyruvate and fumarate release (Figure 4 and S6). The level of

321

Ga-activated citrate exudation was two-fold increased as compared to the control

322

(Figure 4), which is also found in Al toxicity.46 In the presence of Al, citrate and

323

malate exudation increased three- and two-fold in roots.

324

To explore the possible effects of citrate on Ga tolerance, seedlings were

325

exposed to Ga (250 and 500 µM) medium containing different concentrations of

326

citrate (20, 100, 200 and 400 µM) (Figure 5A-C and S7). Exogenous citrate at 20 µM

327

was sufficient and the most effective for alleviating Ga toxicity symptoms. Thus,


Page 14 of 29

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328

exogenous citrate may greatly increase Ga tolerance, probably by the formation of

329

Ga(III)-citrate complexes, which are less toxic or absorbed by plant roots more slowly

330

than the free Ga3+ ion. Accordingly, the secretion of citrate is one of the

331

Ga-detoxification mechanisms of A. thaliana.

332

To further investigate whether supplying citrate decreased Ga uptake in A.

333

thaliana, we quantified Ga concentration in roots and shoots. In 250 µM Ga in

334

medium, the accumulation of root Ga was markedly decreased in the presence of

335

citrate, and shoot Ga concentrations were similar. Thus, citrate inhibits total Ga

336

uptake. With 500 µM Ga, the accumulation of Ga in shoots was positively associated

337

with the concentration of citrate added, although root Ga accumulation was reduced

338

with increasing citrate concentration (Figure 5D-E and S8). In addition, the induction

339

effect of Ga on the expression of MATE and ALMT1 was diminished with exogenous

340

citrate (Figure 5F-G). Hence, exogenous citrate in some ways facilitates Ga

341

detoxification and accelerates root-to-shoot translocation. In other words, plants can

342

accumulate Ga to a higher level in the shoot than root without toxicity (Figure S8).

343

The Ga–citrate complexes may be nonabsorbable or taken up slower than the Ga ion

344

in the root. A similar strategy was observed in the secretion of nicotianamine for zinc

345

tolerance.36 Blocking the entry of excess amounts of metals into plant roots could be a

346

resistance mechanism that enables plants to withstand toxic levels of Ga.

347

Environmental implications. The results of this study showed the mechanisms

348

of Ga toxicity as well as the physiological and molecular basis for Ga resistance in

349

plants. Given the widespread and increasing use of this intermetallic element, it could

350

have

351

contamination. In this study, the exogenous application of citrate to medium

352

containing toxic levels of Ga protected root growth and reduced Ga toxicity. The

potential ecological and human health impacts through


food-chain


353

effectiveness of citric acid in alleviating Ga toxicity may be useful for the chemical

354

manipulation of plants for phytoremediation. However, further research on the

355

physiological and molecular responses of Ga exposure and its possible resistance

356

mechanism in plants will facilitate a mechanistic understanding that is necessary for

357

risk assessment.

358 359


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360

Table 1. Linear combination fitting results for XANES spectra for Ga in plants

No. of standard

Proportion of standards

compounds

in the sample (%)

Ga treatment Sample 500 µM

Ga-citrate

GaOH3

R factor†

Root

2

47.8 (0.132)

52.2 (0.042)

0.031

Shoot

2

68.0 (0.026)

32.0 (0.037)

0.036

361

† The R factor was generated by Athena software for evaluating goodness of a fit.

362

The lower the value, the better the fit.

363 364



Page 18 of 29

365 366

Figure 1. Ga exposure conditions and phenotype characterization of Ga-treated A.

367

thaliana plants. (A) Phenotypes of A. thaliana exposed to 6-750 µM Ga(NO3)3. Bar =

368

1 cm. Seven-day-old A. thaliana seedlings grown in half-strength Murashige and

369

Skoog (½ MS) medium were treated with Ga(NO3)3 for 8 d. (B) Root length. The

370

dashed line represents the root length before Ga treatments; Fresh biomass of (C)

371

roots and (D) shoots of seedlings grown under different Ga levels as in (A).

372

means are averaged from four replicates. Values are presented as mean ± SEM. Bars

373

with different letters are significantly different at p < 0.05.

374 375 376 377


The

Page 19 of 29


378 379

Figure 2. Electron micrographs illustrating the microlocalisation of Ga in

380

Ga-contaminated root cells. A. thaliana was grown on media containing 500 µM Ga.

381

Inset, detail of the granular deposits in intercellular space. Spectra from point of

382

interest (white arrows on photographs) obtained with energy-dispersive X-ray (EDX)

383

analysis demonstrate Ga and co-localized element distribution in contaminated cells:

384

(a), (d) intercellular space, (b) vacuole and (c) cytoplasm. cw: cell wall.

385



386 387

Figure 3. Ga K-edge EXAFS spectra of samples (dashed lines) and the results of the

388

linear combination fitting (LCF; solid lines in red and green).

389


Page 20 of 29

Page 21 of 29


390 391

Figure 4. (A) Citrate and (B) malate concentrations in root exudates of A. thaliana

392

grown under control (con) conditions and 500 µM Ga(NO3)3 (Ga), and 500 µM AlCl3

393

(Al). Plants at 21 d old were hydroponically grown and transferred to treatments for 2

394

d. The means are averaged from four replicates. Values are presented as mean ± SEM.

395

Bars with different letters are significantly different at p < 0.05.

396



397 398

Figure 5. Exogenous citrate affects Ga tolerance in A. thaliana. (A) Phenotypes of

399

plants grown under 250 or 500 µM Ga supplemented with the indicated

400

concentrations of citrate. Seven-day-old seedlings grown in ½ MS medium were

401

treated for 8 d. Bar = 1 cm. (B) Root length and (C) fresh biomass of seedlings grown

402

under Ga treatments. Concentrations of Ga in (D) roots and (E) shoots of seedlings

403

grown under different Ga levels as in (A). qRT-PCR of mRNA levels of (F) AtMATE

404

and (G) AtALMT1 relative to ubiquitin-conjugating enzyme 21 (UBC21) in A.


Page 22 of 29

Page 23 of 29


405

thaliana roots. Root samples were collected from 7-d-old seedlings grown under 250

406

or 500 µM Ga with the indicated concentrations of exogenous citrate for 3 d. The

407

means are averaged from four replicates. Values are presented as mean ± SEM. Bars

408

with different letters are significantly different at p < 0.05.

409 410



411

Acknowledgements

412

This work was financially supported by Academia Sinica, Ministry of Science

413

and Technology (MOST 103-2013-B-002-024-MY3) and Ministry of Education

414

(under the ATU plan), Taiwan. The authors are grateful to Drs. Jyh-Fu Lee and

415

Jeng-Lung Chen for their assistance in XAS measurements at the National

416

Synchrotron Radiation Research Center. We also thank the Imaging Core Facility in

417

the Institute of Cellular and Organismic Biology at Academia Sinica for EM technical

418

support and the Metabolomics Core Facility of the Agricultural Biotechnology

419

Research Center at Academia Sinica for the MS analysis.

420 421


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422

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85x47mm (300 x 300 DPI)


Effect of Gallium Exposure in Arabidopsis thaliana is Similar to

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