Running head: Arsenic speciation in phloem and xylem · PDF fileResearch category: Environmental Stress and Adaptation Plant Physiology Preview. ... The purity of phloem and xylem

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Running head: Arsenic speciation in phloem and xylem exudates 1

Corresponding author: Fang-Jie Zhao 2

Address: Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK 3

Phone: 44 1582 763133 4

Email: [email protected] 5

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Research category: Environmental Stress and Adaptation 7

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Plant Physiology Preview. Published on September 24, 2010, as DOI:10.1104/pp.110.163261

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Arsenic speciation in phloem and xylem exudates of Ricinus communis 9

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Wen-Ling Ye, B. Alan Wood, Jacqueline L. Stroud, P. John Andralojc, Andrea Raab, 11

Steve P. McGrath, Jörg Feldmann, Fang-Jie Zhao* 12

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1. School of Earth and Space Sciences, University of Science and Technology of China, Hefei, 14

Anhui 230026, China (W.-L.Y.) 15

2. Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, United Kingdom (W.-L.Y., J. 16

L.S., P.J.A., S.P.M., F.-J.Z.) 17

3. College of Physical Science, Trace Element Speciation Laboratory (TESLA) Chemistry, 18

University of Aberdeen, Meston Building, Aberdeen AB24 3UE, United Kingdom (B. A.W., 19

A.R., J.F.) 20

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Footnotes: 22

Rothamsted research is an institute of the Biotechnology and Biological Sciences Research 23

Council of the United Kingdom. 24

* Corresponding author: Fang-Jie Zhao (Email: [email protected]) 25

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Abstract 27

How arsenic (As) is transported in phloem remains unknown. To help answer this question, 28

we quantified the chemical species of As in phloem and xylem exudates of Ricinus communis 29

exposed to arsenate [As(V)], arsenite [As(III)], monomethylarsonic acid [MMA(V)] or 30

dimethylarsinic acid [DMA(V)]. In the As(V)- and As(III)-exposed plants, As(V) was the 31

main species in xylem exudate (55 - 83%) whereas As(III) predominated in phloem exudate 32

(70 – 94%). The ratio of As concentrations in phloem to xylem exudate varied from 0.7 to 3.9. 33

Analyses of phloem exudate using high-resolution ICP-MS and accurate mass ES-MS coupled 34

to HPLC identified high concentrations of reduced and oxidized glutathione and some 35

oxidized phytochelatin (PC2), but no As(III)-thiol complexes. It is thought that As(III)-thiol 36

complexes would not be stable in the alkaline conditions of phloem sap. Small concentrations 37

of oxidized glutathione and oxidized PC2 were found in xylem exudate, where there was also 38

no evidence of As(III)-thiol complexes. MMA(V) was partially reduced to MMA(III) in roots, 39

but only MMA(V) was found in xylem and phloem exudate. Despite the smallest uptake 40

among the four As species supplied to plants, DMA(V) was most efficiently transported in 41

both xylem and phloem, and its phloem concentration was 3.2 times that in xylem. Our results 42

show that free inorganic As, mainly As(III), was transported in the phloem of R. communis 43

exposed to either As(V) or As(III), and that methylated As species were more mobile than 44

inorganic As in the phloem. 45



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INTRODUCTION 46

47

Arsenic (As) is an environmental and food chain contaminant that has attracted much 48

attention in recent years. Soil contamination with As may lead to phytotoxicity and reduced 49

crop yield (Panaullah et al., 2009). Food crops are also an important source of inorganic As, a 50

class-one carcinogen, in humans dietary intake, and there is a need to decrease the exposure to 51

this toxin (European Food Safety Authority, 2009). Paddy rice (Oryza sativa) is particularly 52

efficient in As accumulation, which poses a potential risk to the population based on a rice 53

diet (Meharg et al., 2009; Zhao et al., 2010a). Other terrestrial food crops generally do not 54

accumulate as much As as paddy rice; however, where soils are contaminated, relatively high 55

concentrations of As in wheat (Triticum aestivum) grain have been reported (Williams et al., 56

2007; Zhao et al., 2010b). On the other hand, some fern species in the Pteridaceae family are 57

able to tolerate and hyperaccumulate As in the above-ground part to >1000 mg kg-1 dry weight 58

(e.g. Ma et al., 2001; Zhao et al., 2002); these plants offer the possibility for remediation of 59

As-contaminated soil or water (Salido et al., 2003; Huang et al., 2004). A better understanding 60

of As uptake and long-distance transport, metabolism and detoxification is needed for 61

developing strategies for mitigating As contamination, through either decreased As 62

accumulation in food crops or enhanced As accumulation for phytoremediation. 63

The pathways of As uptake by plant roots differ between different As species; arsenate 64

[As(V)] enters plant cells via phosphate transporters, whereas arsenite [As(III)] is taken up via 65

some aquaporins (reviewed by Zhao et al., 2009). In rice, a silicic acid efflux protein also 66

mediates As(III) efflux towards stele for xylem loading (Ma et al., 2008). Methylated As 67

species, such as monomethylarsonic acid (MMA(V)) and dimethylarsinic acid (DMA(V)), 68

which may be present in the environment as products of microbial or algal methylation of 69

inorganic As or from past uses of methylated As pesticides, are taken up by rice roots partly 70

through the aquaporin NIP2;1 (also named Lsi1) (Li et al., 2009). Once inside plant cells, 71

As(V) is reduced to As(III), possibly catalyzed by arsenate reductase(s) such as the plant 72

homologues of the yeast ACR2 (Bleeker et al., 2006; Dhankher et al., 2006; Ellis et al., 2006; 73

Duan et al., 2007). As(III) has a high affinity to thiol (-SH) groups and is detoxified by 74

complexation with thiol-rich phytochelatins (PCs) (Pickering et al., 2000; Schmöger et al., 75

2000; Raab et al., 2005; Bluemlein et al., 2009; Liu et al., 2010). As(III)-PC complexation in 76

roots was found to result in reduced mobility for efflux and for long-distance transport, 77



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possibly because the complexes are stored in the vacuoles (Liu et al., 2010). Excess As(III) 78

causes cellular toxicity by binding to the vicinal thiol groups of enzymes, such as the plastidial 79

lipoamide dehydrogenase, which has been shown to be a sensitive target of As toxicity (Chen 80

et al., 2010). The As hyperaccumulating Pteris species differ from non-hyperaccumulating 81

plants because of enhanced arsenate uptake (Wang et al., 2002; Poynton et al., 2004), little 82

As(III)-thiol complexation (Zhao et al., 2003; Raab et al., 2004) and efficient xylem loading of 83

As(III) (Su et al., 2008). Recently, an As(III) efflux transporter, PvACR3, has been found to 84

play an important role in As(III) detoxification by transporting As(III) into vacuoles in Pteris 85

vittata (Indriolo et al., 2010). 86

With the exception of As hyperaccumulators, most plant species have a limited root-87

to-shoot translocation of As (Zhao et al., 2009). The chemical species of As in xylem exudate 88

have been determined in a number of plant species. As(III) was found to be the predominant 89

species (80 – 100%) in the xylem sap of rice, tomato (Lycopersicon esculentum), cucumber 90

(Cucumis sativus) and P. vittata even when these plants were fed As(V) (Mihucz et al., 2005; 91

Xu et al., 2007; Ma et al., 2008; Su et al., 2010), suggesting that As(V) is reduced in roots 92

before being loaded into the xylem. In other plant species, such as Brassica juncea 93

(Pickering et al., 2000), wheat (Triticum aestivum) and barley (Hordeum vulgare) (Su et al., 94

2010), As(V) accounted for larger proportions (40 – 50%) of the total As in the xylem sap. 95

Studies using HPLC-ICP-MS coupled with electrospray mass spectrometry (ES-MS) showed 96

no evidence of As(III)-thiol complexation in the xylem sap of sunflower (Helianthus annuus) 97

(Raab et al., 2005). When rice plants were exposed to MMA(V) or DMA(V), both As species 98

were found in the xylem sap (Li et al., 2009). Generally, methylated As species are taken up 99

by roots at slower rates than inorganic As, but they are more mobile during the xylem 100

transport from roots to shoots (Marin et al., 1992; Raab et al., 2007; Li et al., 2009). 101

It has been shown that phloem transport contributes substantially to As accumulation 102

in rice grain (Carey et al., 2010). However, little is known about how As is transported in 103

phloem (Zhao et al., 2009). There are no reports on the chemical species of As in phloem 104

exudate. The speciation of As in phloem is important because it dictates how As is loaded in 105

the source tissues and unloaded in the sink tissues, such as grain. Questions with regard to the 106

oxidation state, methylation and complexation of As in phloem sap remain to be answered. 107

Unlike xylem sap, phloem sap is much more difficult to obtain in sufficient quantities for 108

analysis. In the present study, we investigated As speciation in phloem and xylem exudates of 109

Ricinus communis (castor bean), which is widely used as a model plant to investigate phloem 110



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transport of solutes (e.g. Hall et al., 1971; Hall and Baker, 1972; Allen and Smith, 1986; 111

Bromilow et al., 1987). 112

113

RESULTS 114

115

Sugar composition in phloem and xylem exudates of R. communis 116

The purity of phloem and xylem exudates was checked by analyzing the sugar 117

composition. Phloem exudate contained high concentrations of sucrose with the values similar 118

to those reported before (Hall and Baker, 1972; Smith and Milburn, 1980), whilst reducing 119

sugars were absent (fructose) or present at only trace level (glucose) (Table 1). Xylem exudate 120

contained very small concentrations of sucrose, fructose and glucose. These compositions are 121

characteristic of phloem and xylem saps, suggesting little cross contamination. 122

123

Arsenic speciation in R. communis exposed to As(V) or As(III) 124

In the first experiment, 18-d old plants were exposed to 10 μM As(III) or As(V) for 24 125

– 48 h. Total As concentration in phloem exudate increased slightly from 24 to 48 h after As 126

exposure, from approximately 0.8 to 0.9 – 1.2 μM (Fig. 1a). The concentrations were not 127

significantly different between the As(V) and As(III) treatments at either sampling times. The 128

majority of As in phloem exudates was As(III), which accounted for 67 – 79% and 86 – 90% 129

of the total As at 24 and 48 h, respectively, and the remainder was As(V). Again, there was no 130

significant difference in this percentage between the As(V) and As(III) treatments. Xylem 131

exudate was collected after 48 h exposure. Total As concentration in the xylem exudate was 132

about half of that in the phloem exudate (Fig. 1b), with As(III) accounting for 44% in both 133

treatments and the rest being As(V). 134

Analysis of the plant samples collected after 48 h exposure showed much higher As 135

concentrations in roots than in leaves (Fig. 1c, d). 79% of the As in roots was As(III), 136

compared with 96% in leaves, and there was no significant difference in this percentage 137

between the As(III) and As(V) treatments. The sum of all As species detected in roots was 138

smaller in the As(V) than in the As(III) treatment (p = 0.056), but the opposite was observed 139



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for the concentrations in leaves (p < 0.01). Total As concentrations were also determined by 140

acid digestion followed by ICP-MS determination. The sum of As species determined by 141

extraction with a phosphate buffer solution and HPLC-ICP-MS analysis represented 81% and 142

43% of the total As in roots and shoots, respectively. 143

A considerable proportion (43% at 24 h) of As(III) in the nutrient solution was 144

oxidized to As(V), whereas 9% of the As in the As(V) treatment was reduced to As(III) (data 145

not shown). It has been shown that plant roots extrude As(III) to the external medium 146

following As(V) uptake and reduction (Xu et al. 2007). Conversely, As(III) in the medium 147

may be oxidized to As(V) because the nutrient solution was continuously aerated. The partial 148

inter-conversion of As species in the medium may partly explain the lack of significant 149

differences between the As(V) and As(III) treatments. 150

151

Arsenic speciation in R. communis exposed to increasing concentration of As(V) 152

Total As concentration in phloem exudate collected 24 h after As(V) exposure 153

increased with the increasing As(V) concentration in the nutrient solution (p < 0.001; Fig. 2a). 154

The proportion of As(III) varied little (84 – 90%) in the 2 – 50 μM As(V) treatment, but 155

decreased to 71% in the 100 μM As(V) treatment. Xylem exudate could only be collected 156

from the low As(V) treatments (2 – 10 μM) and from one of the four replicates of the 50 μM 157

As(V)-treated plants; As toxicity at high As(V) concentrations might have affected water 158

uptake and/or xylem exudation. As(V) was the main As species in the xylem exudate, 159

accounting for 67% and 79% of the total As in the 2 and 10 μM treatments, respectively, with 160

the rest being As(III) (Fig. 2b). The ratio of phloem to xylem As concentrations was 3.9 and 161

0.7 in the 2 and 10 μM treatments, respectively. No As species were detected in either xylem 162

or phloem exudate from the control plants in the 0 μM As(V) treatment. 163

Arsenic concentrations in roots and leaves increased with the external As(V) 164

concentrations (Fig. 2c, d). Most of the As in leaves (96%) was As(III) and the proportion was 165

not significantly affected by the concentration of As(V) in the medium. In contrast, the 166

proportion of As(III) in roots decreased progressively with the concentration of As(V) 167

exposure, from 81% at 2 μM to 36% at 100 μM As(V). In fact, whilst the concentration of 168

As(V) in roots increased with the As(V) exposure concentration, that of As(III) did not 169

increase beyond the 10 μM As(V) treatment. In this experiment, the extraction and As 170



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speciation method recovered on average 84% and 59% of the total As in roots and leaves, 171

respectively. 172

173

Phosphate supply affects As concentration but not speciation in phloem and xylem 174

exudates of R. communis 175

In this experiment, R. communis plants were exposed to 10 μM As(V) with either 10 or 176

200 μM phosphate. Phloem exudate was dominated by As(III), which accounted for 89 - 94% 177

of the total As at 24 h and 48 h, and the level of phosphate supply had no significant effect on 178

this percentage (p > 0.80) (Fig. 3a). Increasing phosphate from 10 to 200 μM did significantly 179

decrease the concentration of As(III) in phloem exudate, by 50% (p < 0.001) and 26% (p < 180

0.05) at 24 and 48 h, respectively. In contrast, xylem exudate was dominated by As(V), with 181

As(III) accounting for only 17 – 23% of the total As (Fig. 3b). The concentrations of As(V) 182

and As(III) in xylem exudate were decreased by 40% (p < 0.05) and 60% (p < 0.01), 183

respectively, by increasing phosphate concentration in the medium. The percentage of As(III) 184

was not significantly affected. At 48 h, total As concentrations in phloem exudate were 15 – 185

60% higher than those in xylem exudate. 186

187

No evidence of As(III)-thiol complexation in the phloem and xylem exudates of R. 188

communis 189

Phloem and xylem exudates collected after 48 h exposure to 10 μM As(V) were 190

analyzed for As-thiol complexes and free thiol compounds using reverse phase HPLC-high 191

resolution ICP-MS / high resolution ES-MS. The use of high resolution ICP-MS allows 192

quantification of S (m/z 32), which is not possible with normal ICP-MS due to the polyatomic 193

interference of O2 (m/z 32), in addition to simultaneous quantification of As (m/z 75). In 194

addition, the use of high resolution ES-MS allows molecular identification of the compounds. 195

In both phloem and xylem exudate samples, As was eluted in the void volume corresponding 196

to inorganic As; also eluted was sulfate. No As-thiol complexes were detected in either 197

exudate (Fig. 4a, b). High concentrations of reduced and oxidized glutathione (GSH and 198

GSSG) were found in the phloem exudate, as well as some oxidized phytochelatin (PC2) 199

(Table 2). GSSG and oxidized PC2 were detected in the xylem exudate, but at much lower 200



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concentrations than those in the phloem exudate. No GSH was present in the xylem exudate. 201

Inorganic S (sulfate) was abundant in both exudates. 202

203

Methylated As has a greater mobility in phloem and xylem than inorganic As 204

In this experiment, As speciation and the relative mobility were compared among 205

plants exposed to 10 μM As(V), MMA(V) or DMA(V). Only inorganic As species were found 206

in the phloem exudate from the As(V)-treated plants, with As(III) being the predominant 207

species (88% and 92% at 24 h and 48 h, respectively) (Fig. 5a). In the MMA(V)-treated plants, 208

MMA(V) was the only species detected in the phloem exudate, whilst only DMA(V) was 209

found in the DMA(V)-treated plants. Arsenic concentrations in phloem exudate were similar 210

in both 24 h and 48 h samples. Among the three treatments, As concentration in phloem 211

exudate was the highest in the DMA(V) treatment (p < 0.001; Fig. 5a). 212

In xylem exudate, total As concentration followed the order of treatment of MMA(V) 213

< As(V) < DMA(V) (Fig. 5b). The xylem exudate from the As(V)-treated plants contained 214

38% and 62% As(III) and As(V), respectively. The xylem exudate from the MMA(V)-treated 215

plants contained 67% MMA(V), 15% As(III) and 18% As(V). However, the MMA(V) stock 216

solution used in the experiment contained a small amount (~3%) of As(V) as an impurity, 217

which likely explains the occurrence of As(V) and As(III) in the xylem exudate from the 218

MMA(V)-treated plants. Only DMA(V) was found in the xylem exudate of the DMA(V)-219

treated plants. The ratios of As concentration in phloem exudate to that in xylem exudate were 220

2.3, 7.3 and 3.2 in the As(V), MMA(V) and DMA(V) treatments, respectively. 221

The concentrations and species of As in roots and leaves differed between the three 222

treatments (Fig. 5c, d). Similar to the experiments described above, As(III) was the dominant 223

species in roots (87%) and leaves (91%) of the As(V)-treated plants, with As(V) being the 224

minor As species. Arsenic speciation in the roots of the MMA(V)-treated plants was more 225

complex, showing the presence of MMA(V), MMA(III), As(III) and As(V) with percentages 226

of 60, 14, 23 and 3%, respectively (Supplemental Fig. 1). In the leaves of this treatment, As 227

species comprised 89% and 11% MMA(V) and As(III), respectively. In the plants exposed to 228

DMA(V), only DMA(V) was found in both roots and leaves. The MMA(V)-treated plants had 229

smaller (by about 75%) concentrations of total As (sum of all As species) in roots and leaves 230



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than the As(V)-treated plants. In the DMA(V)-treated plants, total As concentrations in roots 231

and leaves were 2% and 61%, respectively, of those in the As(V)-treated plants. 232

In this experiment, the sum of As species accounted for 40 – 60% of the total As 233

concentration by acid digestion, with the exception of a complete extraction (105%) in the 234

DMA(V)-treated shoots. Based on the total As concentrations by acid digestion, uptake of As 235

during the 48 h exposure were 358, 80 and 8.5 nmol g-1 root fresh weight in the As(V), 236

MMA(V) and DMA(V) treatments, respectively. 237

238

DISCUSSION 239

240

The present study was motivated by the gap of knowledge of As speciation in phloem 241

exudate, which has important implications for how As is redistributed around the plant, 242

especially towards the seeds (Zhao et al., 2009). R. communis plants were exposed to four 243

different As species, and As speciation in phloem exudate was compared to that in xylem 244

exudate and roots and leaves. 245

In the plants exposed to either As(V) or As(III), As(III) was found to be the 246

predominant As species in phloem exudate and As(V) the minor species. The proportion of 247

As(III) varied somewhat among different experiments and was also influenced by the 248

concentrations of As(V) in the external solution, but generally fell within the range of 80 – 249

90% (Figs 1, 2, 3, 5). The proportion dropped to around 70% when plants were exposed to a 250

high concentration (100 μM) of As(V). Furthermore, inorganic As in the phloem exudate was 251

not complexed with thiol compounds, even though the concentrations of GSH, GSSG and PC2 252

far exceeded that of inorganic As (Fig. 4 and Table 2). The lack of thiolated arsenic is not an 253

analytical artefact, because the extraction and the chromatographic separation has been 254

validated by using XANES/EXAFS of plant tissues directly (Bluemlein et al., 2008). This lack 255

of thiol complexation may be explained by the high pH of phloem sap in R. communis (7.4 – 256

8.2) (Hall and Baker, 1972; Allen and Smith, 1986). It is known that As(III) forms stable 257

complexes with thiol compounds under acidic conditions but not in neutral-alkaline 258

environments (Schmöger et al., 2000). A number of As(III)-thiol complexes have been 259

identified in roots and shoots of different plant species (Raab et al., 2004; Raab et al., 2005; 260

Liu et al., 2010), whilst X-ray absorption studies indicate that most of As(III) in non-261



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hyperaccumulating plant species is bonded with S (Pickering et al., 2000; Dhankher et al., 262

2002). As(III)-thiol complexes are likely to be stored in vacuoles where the acidic pH favors 263

the stability of the complexes. Increasing As(III)-thiol complexation was found to decrease 264

xylem transport of As(III) (Liu et al., 2010), and may also affect As(III) transport in phloem in 265

the same way. Given that uncomplexed As(III) or at least only weakly complexed As(III), 266

which is not detectable by chromatographic methods (Feldmann et al., 2009), and, to a smaller 267

extent, As(V), are the only species of As in phloem exudate, transporters for these species are 268

likely to be involved in the loading and unloading during phloem transport, e.g. some of the 269

aquaporins, Lsi2-like or ACR3-like proteins for As(III) and phosphate transporters for As(V). 270

Similar to our study, thiol compounds (mainly glutathione and PC2) were found to be 271

abundant in the phloem sap from Brassica napus exposed to Cd (Mendoza-Cózatl et al., 2008). 272

Experiments with grafting and shoot-specific expression of the genes involved in glutathione 273

synthesis have shown that glutathione and PCs are transported via phloem from shoots to 274

roots of Arabidopsis thaliana (Chen et al., 2006; Li et al., 2006). Unlike As, it is possible that 275

Cd-PC complexes are transported in phloem (Mendoza-Cózatl et al., 2008), because these 276

complexes are stable in the slightly alkaline conditions of phloem sap, but not in acidic 277

environments (Johanning and Strasdeit, 1998). 278

Different from phloem exudate, more As(V) than As(III) was found in xylem exudate 279

of R. communis, with the former typically accounting for 60 - 80% of the total As (Figs. 1, 2, 3 280

and 5). There was also no evidence of As(III)-thiol complexes (Fig. 4), even though xylem sap 281

pH (~5.0) in R. communis (Allen and Smith, 1986) is favorable to the stability of As(III)-thiol 282

complexes. Small concentrations of GSSG and oxidized PC2 were found in xylem sap of R. 283

communis (Table 2), which was consistent with a previous study on sunflower (Raab et al., 284

2005). The study of Raab et al. (2005) also found no evidence of As(III)-thiol complexation in 285

the xylem sap of sunflower. The presence of only low concentrations of non-reactive 286

(oxidized) thiols in xylem sap probably explains the lack of As(III)-thiol complexation. 287

Similarly, Mendoza-Cózatl et al. (2008) found small concentrations of glutathione and traces 288

of PC2 in the xylem sap of Brassica napus exposed to Cd, although the analytical method used 289

did not separate oxidized from reduced thiols. 290

Previous studies have shown that As(III) dominates in the xylem sap collected from a 291

number of plant species exposed to As(V), including tomato, rice, cucumber and the As 292

hyperaccumulator Pteris vittata (Mihucz et al., 2005; Xu et al., 2007; Su et al., 2008; Su et al., 293



13

2010). In contrast, As(V) was found to be an important As species in the xylem sap collected 294

from Brassica juncea, Holcus lanatus and wheat, accounting for between 30 – 60% of the 295

total As (Pickering et al., 2000; Su et al., 2010). The relative percentages of As(III) and As(V) 296

in xylem sap may depend on a number of factors, such as the capacity of arsenate reduction in 297

roots, the abundance and the relative affinities of As(III) or As(V) transporters involved in 298

xylem loading, the degree of As(III)-thiol complexation in roots and the presence of 299

competing ions. There is evidence that more As(V) was loaded into xylem when the arsenate 300

reduction capacity in roots was exceeded in the treatments with high As(V) concentrations 301

(Fig. 2); this also had the effect of increasing As(V) percentage in the phloem exudate. 302

Complexation of As(III) with thiol compounds in roots may decrease As(III) loading into the 303

xylem (Liu et al. 2010), resulting in a larger As(V)% in the xylem exudate than that in the root 304

tissues. This explanation may also apply to the higher As(V)% in phloem sap than in leaves. 305

Phosphate is known to inhibit As(V) uptake (Ullrich-Eberius et al., 1989; Abedin et al., 2002; 306

Wang et al., 2002), and may also inhibit As(V) loading into xylem or phloem. However, this 307

latter effect was not observed in our experiment (Fig. 3). Increasing phosphate supply 308

decreased As concentrations in both xylem and phloem without significantly affecting the 309

percentages of the two As species, suggesting that phosphate inhibits the influx of arsenate 310

into roots rather than its loading into xylem. Comparing xylem and phloem exudates, the 311

concentrations of As in the latter were, in most cases, higher than those in the former. The fact 312

that both leaves and phloem exudate contained much higher proportions of As(III) than xylem 313

exudate indicates that As(V) is reduced to As(III) in leaves as well as in roots. 314

Previous studies have shown that methylated As species are generally taken up more 315

slowly than inorganic As, but translocated more efficiently from roots to shoots (Marin et al., 316

1992; Abedin et al., 2002; Raab et al., 2007; Li et al., 2009). Similar results were obtained 317

with R. communis, with As uptake following the order of As(V) > MMA(V) > DMA(V). In R. 318

communis roots, MMA(V) was partly reduced to MMA(III), although only MMA(V) was 319

loaded into the xylem and phloem (Fig. 5, Supplemental Fig. 1). According to the Challenger 320

pathway of As methylation established in microorganisms (Bentley and Chasteen, 2002), 321

reduction of MMA(V) to MMA(III) is a prerequisite step before further addition of the methyl 322

group. However, no DMA(V) was produced in the MMA(V)-treated plants, indicating no 323

further methylation of MMA(III) in R. communis within the 48 h exposure period. Despite a 324

very limited uptake of DMA(V), its concentration was the highest in both xylem and phloem 325

exudates among the three As treatments, with the phloem concentration being about 3 times 326



14

the xylem concentration (Fig. 5). MMA(V) concentrations in phloem exudate were also 327

comparable to or larger than inorganic As despite smaller uptake of the former species. These 328

results indicate that methylated As, particularly the dimethyl species, are more mobile than 329

inorganic As in the phloem. Consistent with this finding, Carey et al. (2010) showed that 330

DMA(V) was transported to rice grain much more efficiently than inorganic As. Uptake of 331

undissociated MMA(V) and DMA(V) by rice roots is partly mediated by the aquaporin 332

NIP2,1 (Li et al., 2009), but membrane transporters responsible for loading and unloading of 333

methylated As species in phloem remain unknown. It has been suggested that a lack of thiol 334

complexation of MMA(V) and DMA(V) may explain their high mobility in xylem transport 335

(Raab et al., 2007); this explanation may also apply to the high mobility in phloem observed in 336

the present study. Increased lipophilicity with methylation may be another reason, although 337

this is apparently associated with decreased uptake by roots. 338

The identification of the forms of As in phloem exudate should help future research to 339

elucidate the mechanisms of As loading and unloading in the phloem. Future work should also 340

investigate if the forms of As transported in phloem are similar in food crops such as rice. 341

342

MATERIALS AND METHODS 343

344

Plant culture 345

Seeds of R .communis var. Gibsonii were surface sterilized by immersing them in 0.5% 346

NaOCl for 15 min. After rinsing, seeds were soaked in deionized water overnight and then 347

germinated in moist vermiculite. After germination, seedlings were grown in hydroponic 348

culture for 18 d with a modified ½ strength Hoagland nutrient solution (1 seedling per 1-L 349

pot). The composition of the nutrient solution were 3 mM KNO3, 2 mM Ca(NO3)2, 0.5 mM 350

NH4H2PO4, 1 mM MgSO4, 2 mM MES (2-morpholinoethanesulfonic acid; pH adjusted to 6.0 351

with KOH), 100 µM Fe(III)-EDTA, 0.55 µM MnCl2, 46 µM H3BO3，9 µM MnCl2，0.75 µM 352

ZnSO4 and 0.35 µM CuSO4. The nutrient solution was aerated continuously and renewed once 353

every 3 d. The growth conditions were 16 h photoperiod with a light intensity of 350 µmol m-354

2s-1, day : night temperatures 28:25ºC and 70% relative humidity. After 18-d growth, the fresh 355

weight of shoots and roots were on average 30 and 18 g per plant. 356



15

357

Arsenic exposure and collection of phloem and xylem exudates 358

A series of experiments were conducted with 18-d old R. communis plants, varying in 359

the species and concentrations of As added to the nutrient solution. The first experiment 360

compared As exposure in the forms of As(V) (Na2HAsO4) or As(III) (NaAsO2) at 10 μM. The 361

second experiment investigated the dose response to increasing As(V) concentrations (0, 2, 10, 362

50 and 100 μM). In the third experiment, plants were exposed to 10 μM As(V) with either 10 363

or 200 μM phosphate. In the fourth experiment, xylem and phloem exudates were collected 364

from plants exposed to 10 μM As(V) for the analysis of thiol compounds and As(III)-thiol 365

complexes. The fifth experiment compared the As mobility and speciation in xylem and 366

phloem exudates after R. communis plants were exposed to 10 μM As(V), MMA(V) or 367

DMA(V). Each treatment was replicated in 3 – 4 pots. During As exposure, the phosphate 368

concentration in the nutrient solution was reduced to 10 μM to allow more uptake of As(V) 369

(except the high P treatment in the third experiment). Phloem exudate was collected at 24 and 370

48 h after As exposure, and xylem exudate at 48 h exposure, except in the second experiment 371

when both exudates were collected at 24 h. To collect phloem exudate, a shallow, V-shaped 372

incision was made into the bark of the stem at about 2 cm above the stem/root junction with a 373

razor blade (Hall et al., 1971). Phloem exudate was collected in a glass capillary tube (50 or 374

100 µl) for 1 h. After collection of phloem exudate, the bark (containing the phloem tissue) 375

was removed from a 30-40 mm section of stem near the base by ring-girdling (Allen and 376

Smith, 1986). One h later, the stem was cut at the top of the ring-girdled section and xylem 377

exudate collected by pipette for 1 h after decapitation. The collected sap samples were stored 378

on ice, and diluted with a phosphate buffer solution (PBS) containing 2 mM NaH2PO4 and 0.2 379

mM Na2-EDTA (pH 6.0) before As speciation analysis. For analysis of thiols and As(III)-thiol 380

complexes, phloem and xylem saps were diluted with 1% formic acid in 1:5 ratio. After 381

collection of phloem and xylem exudates, plant roots were rinsed briefly in an ice-cold 382

desorption solution containing 1 mM K2HPO4, 0.5 mM Ca(NO3)2 and 5 mM MES (pH 6.0), 383

and immersed in 1 L of the same solution for 10 min to remove apoplastic As (Xu et al., 2007). 384

Shoots were rinsed in deionized water. Root and leaf samples were blotted dry, frozen in 385

liquid N2 and were ground to fine powder with a mortar and pestle. Aliquots of nutrient 386

solutions were taken at 24 and 48 h to monitor the changes in As species. 387

388



16

Analysis of sugar composition in xylem and phloem exudates 389

The predominant soluble sugars in both xylem and phloem exudates were identified 390

and quantified by high-pH anion exchange HPLC using a Dionex CarboPac PA1 guard (4 x 391

50 mm) and analytical (4 x 250 mm) column connected to a Dionex DX500 (Sunnyvale, CA, 392

USA), with refrigerated autosampler. Each separation was achieved within 20 min by isocratic 393

elution using 40 mM sodium hydroxide at a flow rate of 1 mL min-1. Peak detection was by 394

pulsed amperometry using a gold working electrode and an Ag/AgCl reference electrode 395

together with the Manufacturer’s electrode potential settings, optimized for carbohydrate 396

detection. The calibration standards were linearly related to detector response over the range 397

employed (0, 1.25, 2.5 and 5 nmols of glucose, fructose and sucrose) and the exudates were 398

diluted as necessary to give component signals which fell within the standard range (xylem 399

20-fold; phloem 2000-fold). An injection volume of 25 µL was used throughout. 400

401

Analysis of As species using anion exchange HPLC-ICP-MS 402

Aliquots (0.2 - 0.5 g) of the ground materials were extracted with 20 ml PBS for 1 h 403

under sonication. The extracts were filtered through 0.45 µm filters. Arsenic speciation in 404

phloem and xylem saps, nutrient solutions and plant extracts were determined using HPLC-405

ICP-MS (Agilent LC1100 series and Agilent ICP-MS 7500ce, Agilent Technologies, Santa 406

Clara, CA, US). Arsenic species [As(III), As(V), DMA(V), MMA(V), and MMA(III)] were 407

separated by an anion-exchange column (Hamilton PRP X-100, Reno, NV, USA) as described 408

previously (Li et al., 2009). The mobile phase was either 6.6 mM NH4H2PO4 and 6.6 mM 409

NH4NO3 (pH 6.3) with a flow rate of 0.6 ml min-1 (method 1), or 2 mM NH4H2PO4 and 2 mM 410

NH4NO3 (pH 6.3) with a flow rate of 0.8 ml min-1 (method 2). Method 1 was used to separate 411

As(III), As(V), MMA(V) and DMA(V), and method 2 As(III), MMA(V), MMA(III) and 412

DMA(V) (Li et al., 2009). The outlet of the separation column was connected to a concentric 413

nebulizer and a water-jacketed cyclonic spray chamber of the ICP-MS. Germanium (Ge) was 414

used as the internal standard which was mixed continuously with the post-column solution 415

through a peristaltic pump. Signals at mass-to-charge ratio (m/z) 75 (As), 72 (Ge), and 35 416

(chlorine [Cl]) were collected with a dwell time of 0.3 s for As and Ge and 0.1 s for Cl. 417

Possible polyatomic interference of 40Ar35Cl on m/z 75 was removed by the Agilent octopole 418

reaction system using helium as the reaction gas. The As signal was normalized by the Ge 419

signal to correct any signal drift during the analysis. Arsenic peaks were identified by 420



17

comparisons with the retention times of standard compounds. Analysis of As species was 421

carried out immediately following sample collection or extraction. For each batch of samples, 422

the analysis was completed within 12 h; repeated analyses showed no changes in As 423

speciation during this period of time. To test the stability of As species in xylem exudate 424

during collection and subsequent analysis, As(III) or As(V) was spiked to the exudate 425

collected from plants untreated with As. No evidence of As(III) oxidation or As(V) reduction 426

was found. 427

428

Analysis of thiols and As(III)-thiol complexes using reverse phase HPLC-high resolution 429

ICP-MS – high resolution ES-MS 430

Analysis of thiols and As(III)-thiol complexes in phloem and xylem saps was carried 431

out using HPLC coupled with high-resolution ICP-MS (Element 2, Thermo Fisher Scientific, 432

Bremen, Germany) and high resolution ES-MS (LTQ Orbitrap Discovery, Thermo Fisher 433

Scientific, Bremen, Germany). Separation was performed on a C18 reverse-phase column, 434

using a water-methanol gradient described previously (Bluemlein et al., 2008; Bluemlein et al., 435

2009; Liu et al., 2010). For the ICP-MS analysis, As and sulfur (S) were measured on m/z 75 436

and 32, respectively. Rhodium (Rh) was added post-column as internal standard and its 437

signal was measured on m/z 103. The ICP-MS was used in medium resolution. The molecular 438

masses of the eluted compounds were determined with high resolution on the ES-MS during a 439

chromatographic run, where glutathione from a xylem sap sample was used as an internal 440

standard for mass determination and correction. A retention time dependent element-specific 441

response using dimethylarsinic acid and methionine was used for quantification of any As and 442

S containing molecules as described in Bluemlein et al. (2008). 443

444

Analysis of total As in plant tissues 445

Ground plant samples were digested in 5 ml high purity HNO3/HClO4 (87/13, v/v). 446

Total As concentrations in the samples were determined by ICP-MS (Agilent 7500ce) 447

operating in the helium gas mode to remove possible interference of 40Ar35Cl on m/z 75. 448

449

Data analysis 450



18

Significance of treatment effects was tested by analysis of variance (ANOVA). Where 451

necessary, data were logarithmically transformed prior to ANOVA. Treatment means were 452

compared using the least significant difference (LSD). 453

454

Supplemental Materials: 455

Figure 1. HPLC-ICP-MS chromatograms of As species in roots, leaves, xylem and phloem 456

exudates of Ricinus communis exposed to 10 μM arsenate, DMA or MMA. 457

458

ACKNOWLEDGEMENT 459

We thank Dr. Richard Bromilow for advice on phloem sap collection. 460

461

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601

602



23

Table 1. The composition of sugars in phloem and xylem exudates of Ricinus communis 603

(mean ± SE, n = 4). 604

Exudate Sucrose (mM)

Glucose (mM)

Fructose (mM)

Phloem 238 ± 15.8 0.12 ± 0.12 0 ± 0

Xylem 1.20 ± 0.32 0.29 ± 0.08 0.36 ± 0.11 605

606



24

Table 2. The concentrations of sulfate, reduced glutathione (GSH) and oxidized glutathione 607

(GSSG) and oxidized PC2 in phloem and xylem exudates of Ricinus communis exposed to 10 608

μM arsenate for 48 h (mean ± SE, n = 2). 609

Exudate

Sulfur species (nmol g-1)

Sulfate GSH GSSG Oxidized PC2

Phloem 141 ± 56 64 ± 33 22 ± 1.4 1.2 ± 0.7

Xylem 131 ± 8 0 ± 0 5.8 ± 0.8 0.2 ± 0.01 610



25

Figure captions: 611

Fig. 1. Arsenic speciation in phloem exudate (a), xylem exudate (b), roots (c) and leaves of 612

Ricinus communis exposed to 10 µM arsenate or arsenite for 24 or 48 h. Error bars are one SE 613

(n = 3 - 4). 614


Ricinus communis exposed to 2 - 100 µM arsenate for 24 h. Error bars are one SE (n = 3 - 4, 616

except the 50 µM treatment in which only one replicate produced xylem exudate; the 100 µM 617

treatment produced no xylem exudate). 618

Fig. 3. Arsenic speciation in phloem exudate (a) and xylem exudate (b) of Ricinus communis 619

exposed to 10 µM arsenate with either 10 µM (low P) or 200 µM phosphate (high P) for 24 or 620

48 h. Error bars are one SE (n = 3 - 4). 621

Fig. 4. Overlaid RP-HPLC chromatograms for the phloem (a) and xylem (b) exudates detected 622

online simultaneously by using ICP-MS (m/z 32 and 75) and ES-MS (m/z 308, 538 and 613, 623

[M+H]+). The sulfur trace (m/z 32) shows the relative quantity of the inorganic sulfur 624

compounds eluting near the void at 2 min compared to GSSG and oxidized PC2. 625


Ricinus communis exposed to 10 µM arsenate, MMA(V) or DMA(V) for 24 or 48 h. Error 627

bars are one SE (n = 3 – 4). 628



Figure 1. Arsenic speciation in phloem exudate (a), xylem exudate (b), roots (c) and leaves of Ricinus communis

exposed to 10 µM arsenate or arsenite for 24 or 48 h. Error bars are one SE (n = 3 - 4).

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

As(III), 24h As(V), 24h As(III), 48h As(V), 48h

Con

cent

ratio

n (μ

M)

PhloemAs(V)

As(III)

0

0.1

0.2

0.3

0.4

0.5

0.6

As(III), 48h As(V), 48h

Con

cent

ratio

n (μ

M)

XylemAs(V)

As(III)

0

50

100

150

200

250

300

As(III) As(V)

Con

cent

ratio

n (n

mol

/g F

W)

Roots

As(V)

As(III)

0

2

4

6

8

10

As(III) As(V)

Con

cent

ratio

n (n

mol

/g F

W)

Leaves

As(V)

As(III)

(c) (d)

(b)(a)

w

ww

.plantphysiol.orgon M

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ll rights reserved.


Figure 2. Arsenic speciation in phloem exudate (a), xylem exudate (b), roots (c) and leaves of Ricinus communis

exposed to 2 - 100 µM arsenate for 24 h. Error bars are one SE (n = 3 - 4, except the 50 µM treatment in which

only one replicate produced xylem exudate; the 100 µM treatment produced no xylem exudate).

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

2 10 50 100

As

conc

entr

atio

n (μ

M)

As(V) (µM)

Phloem

As(V)

As(III)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

2 10 50 100

As

conc

entr

atio

n (μ

M)

As(V) (µM)

Xylem

As(V)

As(III)

0

5

10

15

20

25

30

2 10 50 100

As

conc

entr

atio

n (n

mol

/g F

W)

As(V) (µM)

Leaves

As(V)

As(III)

050

100150200250300350400450

2 10 50 100

As

conc

entr

atio

n (n

mol

/g F

W)

As(V) (µM)

Roots

As(V)

As(III)

(a) (b)

(c) (d)

w

ww



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Figure. 3. Arsenic speciation in phloem exudate (a) and xylem exudate (b) of Ricinus communis exposed to 10 µM

arsenate with either 10 µM (low P) or 200 µM phosphate (high P) for 24 or 48 h. Error bars are one SE (n = 3 - 4).

0.0

0.2

0.4

0.6

0.8

1.0

Low P, 24h High P, 24 h Low P, 48h High P, 48 h

Con

cent

ratio

n (μ

M)

Phloem As(V)

As(III)

0.0

0.2

0.4

0.6

0.8

1.0

Low P, 48h High P, 48 h

Con

cent

ratio

n (μ

M)

Xylem

As(V)

As(III)

(a) (b)

w

ww



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Figure 4. Overlaid RP-HPLC

chromatograms for the phloem (a) and

xylem (b) exudates detected online

simultaneously by using ICP-MS (m/z 32

and 75) and ES-MS (m/z 308, 538 and 613,

[M+H]+). The sulfur trace (m/z 32) shows

the relative quantity of the inorganic

sulfur compounds eluting near the void at

2 min compared to GSSG and oxidized PC2.

Retention time, min

0 10 20 30 40

Intensity, arb

itrary

m/z 75 (As)

m/z 32 (S)

m/z 613 (GSSG)

m/z 538 (ox PC2)

b)

Retention time, min

0 10 20 30 40

Intensity, a

rbitra

ry

m/z 75 (As)

m/z 32 (S)

m/z 308 (GSH)

m/z 613 (GSSG)

m/z 538 (ox PC2)a)

w

ww



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Copyright ©

2010 Am

erican Society of P

lant Biologists. A

ll rights reserved.


0

1

2

3

4

5

As(V) MMA DMA

Con

cent

ratio

n (n

mol

/g F

W) Shoots DMA(V)

MMA(V)

As(V)

As(III)

0

20

40

60

80

100

120

140

As(V) MMA DMA

Con

cent

ratio

n (n

mol

/g F

W) Roots DMA(V)

MMA(III)

MMA(V)

As(V)

As(III)

0.0

0.2

0.4

0.6

0.8

1.0

Con

cent

ratio

n (μ

M) Xylem DMA(V)

MMA(V)

As(V)

As(III)

0

1

2

3

Con

cent

ratio

n (μ

M) Phloem DMA(V)

MMA(V)

As(V)

As(III)

Figure 5. Arsenic speciation in phloem exudate (a), xylem exudate (b), roots (c) and leaves of Ricinus communis exposed

to 10 µM arsenate, MMA(V) or DMA(V) for 24 or 48 h. Error bars are one SE (n = 3 – 4).

(b)

(c) (d)

(a)

w

ww



Dow

nloaded from

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2010 Am

erican Society of P

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ll rights reserved.


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Running head: Arsenic speciation in phloem and xylem · PDF fileResearch category: Environmental Stress and Adaptation Plant Physiology Preview. ... The purity of phloem and xylem