Microchim Acta 151, 141–152 (2005)
DOI 10.1007/s00604-005-0394-8
Review
Arsenic Speciation Governs Arsenic Uptake and Transport
in Terrestrial Plants
Mieke Quaghebeur and Zed Rengel

Soil Science and Plant Nutrition, School of Earth and Geographical Sciences, University of Western Australia,
35 Stirling Highway, Crawley WA 6009, Australia
Received August 13, 2004; accepted May 24, 2005; published online August 22, 2005
# Springer-Verlag 2005
Abstract. Arsenic is a carcinogenic metalloid that
occurs in the environment in a variety of chemical
forms, showing different mobility, bioavailability
and toxicity. Terrestrial plants may accumulate large
amounts of arsenic. To understand how terrestrial
plants take up, transport and metabolise these arsenic
species, it is essential to characterise arsenic speciation
in plant tissues. Given that As species can be trans-
formed from one form to another during sample
preparation and the measurement process, arsenic spe-
ciation in biological extracts needs to be performed
with great care. This paper describes the methods used
to measure arsenic speciation in plant tissue and
assesses the role of As speciation in As metabolism
in higher plants.
Key words: Arsenic; metabolism; metalloid; plant; speciation.
Arsenic contamination of the environmental is a major
concern worldwide. The exploitation of As-containing
groundwater in large areas of Asia causes a life-threat-
ening problem for millions of people via contamina-
tion of drinking water and consumption of food grown
in As-contaminated soil or irrigated with As-contam-
inated water. In most areas, the contamination of

Author for correspondence. E-mail: Zed.Rengel@uwa.edu.au
groundwater occurs naturally because of As-contain-
ing sediments. The As contamination has been
acknowledged as a ‘‘major public health issue’’ by
the World Health Organization (WHO) (1999), with
the requirement to deal with it on an ‘‘emergency
basis’’.
Arsenic can be present in the environment in
various chemical forms (arsenate [As(V)], arsenite
[As(III)], monomethylarsonic acid [MMA], dimethyl-
arsinic acid [DMA], trimethylarsine [TMA], arseno-
choline [AsC], arsenobetaine [AsB], arsenosugars,
etc.). These species have different physical and chem-
ical characteristics, resulting in various degrees of
mobility, bioavailability and toxicity. In general, inor-
ganic arsenicals are more toxic than organic ones;
moreover, arsenicals in the trivalent oxidation state
are more toxic than those in the pentavalent oxidation
state [1, 2]. Therefore, determination of As speciation
is essential for understanding and monitoring the fate
of As in the environment.
The speciation of As is quite complex, with both
organic and inorganic As species occurring in the en-
vironment. Organic As species [MMA, DMA, AsC,
AsB] are predominantly found in marine organisms
(lobsters [3], brown algae [4], seaweed [5], fish [6]
and seawater bivalves [7]). In terrestrial environments,
inorganic As species [As(V) and As(III)] prevail,
although small quantities of organic As species
142 M. Quaghebeur and Z. Rengel

have also been identified, most likely due to either Arsenic Species in the Soil-Plant
microbial activity or the application of As-based Environment
organic pesticides and insecticides.
Different organic and inorganic As compounds have
Arsenic species are toxic to most plant species.
been identified in the soil-plant environment (Fig. 1),
The discovery of As-resistant and As-hyperaccumu-
but the inorganic arsenite [As(III)] and arsenate
lating plant species triggered the interest in under-
[As(V)] are the most common. Because plants absorb
standing the distribution, regulation and metabolism
ions from the soil solution, the concentration and spe-
of As species in terrestrial plants. Hence, the need
ciation of As compounds in the soil solution are of
evolved to establish the capacity for qualitative and
particular interest. Generally, As(V) is the dominant
quantitative determination of As speciation in various
species in the soil solution under oxidising conditions,
plant parts.
whereas As(III) is the prevailing species under mod-
The present paper critically assesses the role of As
erately reducing conditions [8–11].
speciation in understanding As uptake, transport and
The use of organic arsenicals in agriculture and the
metabolism in higher plants. First, we describe the
methylation of inorganic As compounds by microor-
occurrence and properties of different As species in
ganisms can result in the occurrence of organic As
the plant-soil environment, followed by discussing the
compounds in the soil solution. Generally, only small
current understanding about As uptake and As resis-
amounts of MMA, DMA and TMA are formed by
tance in higher plants. Secondly, we evaluate the
microbes in aerobic [12–14] and anaerobic soils
methods for measuring As speciation in plant tissue
[15], although up to 40% DMA was found in the soil
and assess the role of arsenic speciation in As metab-
solution when rice plants were grown under flooded
olism in higher plants.

Fig. 1. Chemical structure of As species detected in terrestrial plants (adapted from [20])
As Speciation Governs Arsenic Uptake and Transport in Terrestrial Plants
conditions [16, 17]. Organic As species in soil may be
converted back to inorganic ones by soil microorgan-
isms [18, 19] or may be lost from soil through vola-
tilisation by reduction of the arsenic species to the
corresponding arsines [11, 12, 16].
Arsenic Uptake by Terrestrial Plants
The level of As in the plants correlates well with the
As concentration in the soil solution [11]. Moreover,
not only the total As concentration in solution, but
also the speciation of As influences As uptake [21].
The chemical behaviour of As(V) is similar to
P(V); hence, As(V) and P(V) may be taken up by
the same plasma membrane transport system. Indeed,
Pauwels et al. [22] found that both ions were taken up
by yeast with similar kinetics and were inhibited by
the same chemicals, whereas in Chlorella sp. and
Lemna gibba (duckweed) As(V) acted as a competi-
tive inhibitor of P(V) uptake [23, 24]. Studies with
Hordeum vulgare (barley) and Holcus lanatus (velvet
grass) found that P(V) and As(V) are taken up by the
same plasma membrane transport uptake system in
terrestrial plants, although the transporters had higher
affinity for P(V) than As(V) [25, 26].
A recent study has shown that undissociated
As(III) (pKa 9.2) is taken up by Saccharomyces
cerevisiae (baker’s yeast) via a glycerol-transporting
channel [27]. These channels transport water and
solutes in bacteria, fungi, plants and animals and are
called aquaporins [28]. The As(III) is also taken up
by these glycerol-transporting channels in Oryza
sativa (rice). Therefore, different plasma membrane
systems are responsible for uptake of As(III) and
As(V) [29].
Because the inorganic arsenic species generally
prevail in the soil-plant environment, only a few stud-
ies examined uptake of organic As species [17, 30–
32]. Uptake of DMA and MMA was generally lower
than uptake of As(III) and As(V) [17, 30–32].
Arsenic Resistance in Higher Plants
The observation that some plant species can grow on
mine spoils that contain high concentrations of As has
led to the assumption that these plant species may
have developed specific resistance mechanisms to
As [33–36]. Plants can achieve metal resistance by
using two types of mechanisms: (i) avoidance by
minimising metal uptake across the plasma membrane
143
of root cells [37], and (ii) tolerance to metals accu-
mulating in the symplasm [38].
Regulation of As Uptake
A number of plant species can avoid As toxicity by
regulating the uptake of As (e.g. Holcus lanatus [26],
Agrostis capillaris, Deschampsia caespitosa [39] and
Cytisus striatus [40]). Phosphate and As(V) are taken
up by the same transporters. At high P(V) concentra-
tions, plants take up P(V) by a low-affinity uptake
system. In contrast, at low P(V) levels, a high affinity
uptake system controlling P(V) uptake is induced
[41]. Meharg and Macnair [26] found that As-resistant
Holcus lanatus was able to suppress the high-affinity
P(V) uptake system. Hence, at low concentration of
P(V) and As(V), uptake of both ions was greater in
non-resistant compared to As-resistant Holcus lanatus
plants.
Root coating with brownish precipitate, indicating
the formation of an iron plaque due to the oxidising
activity of roots, was observed on Aster tripolium and
Oryza sativa plants grown under reducing conditions
[where As(III) was the dominant As chemical form]
[10, 42]. It is possible that As(III) may have been
immobilised due to oxidation to As(V) and subse-
quent sorption onto Fe-oxyhydroxides to coat the
roots. This strategy most likely results in decreased
As uptake and can therefore be considered an As
detoxification strategy.
Methylation of Inorganic As Species
Aquatic organisms and fungi are able to detoxify As
by converting inorganic As species to less toxic
organic As species [43, 44]. It has been proposed that
terrestrial plants detoxify inorganic As species by the
formation of organic As species [45]; however, there
is no clear evidence yet to support this hypothesis.
The small amounts of organic As species identified
in terrestrial plants suggest that it is unlikely that
the methylation of inorganic As would be an impor-
tant As detoxification strategy in higher plants. So far,
it is not even clear whether terrestrial plants have the
capacity to methylate inorganic As species or whether
these organic As species are taken up from the soil or
nutrient solution, where they are formed by microbial
action [20]. Recently, methylated As species that con-
tain As in the trivalent oxidation state have been iden-
tified as intermediate products in the methylation
144
pathway. Although the pentavalent organic arsenic
species are generally less toxic than inorganic As spe-
cies [43, 44], the trivalent organic arsenic species
appear at least as toxic, or somewhat more toxic than
the inorganic species [46, 47]. Hence, the importance
of the biomethylation of As as a detoxification process
has been questioned [47].
Complexation of Inorganic As
to Phytochelatins
Phytochelatins (PC) are heavy-metal-binding peptides
derived from glutathions (GSH) with the general
structure (
-Glu-Cys)n-Gly (n ¼ 2 to 11) [48, 49].
The biosynthesis of phytochelatins involves the trans-
peptidation of
-glutamyl-cysteinyl dipeptides from
GSH by the action of constitutively expressed phyto-
chelatin synthase [50, 51]. Synthesis of phytochelatins
is induced by a range of cations, such as Agþ , Cd2þ ,
Cu2þ , Hg2þ and Pb2þ [48]. As the complexed metals
are less toxic than the free forms, the production of
phytochelatins can decrease the acute toxicity of metal
ions in plants. Recent studies have confirmed that
As(III) is complexed with phytochelatins in a range
of terrestrial plant species [52–55], suggesting that
phytochelatins play an important role in decreasing
the toxicity of As in terrestrial plants.
In vivo and in vitro biosynthesis of As(III)-PC com-
plexes resulted in a stoichiometry of As(III) to phy-
tochelatin-derived Cys residues of approximately 1 to
3 [54]. It was later confirmed by electrospray ioniza-
tion mass spectroscopy that three thiol groups from
two phytochelatin molecules will coordinate one
As(III) molecule [54]. Both As(III) and As(V) trigger
the formation of phytochelatins in plants [50, 54, 56].
In contrast to As(III), however, As(V) has no affinity
for thiol groups; it is therefore unlikely that phyto-
chelatins would complex As(V) [57]. Hence, it is
suggested that once inside the cytoplasm, As(V) is
partially reduced to As(III) (which can then form
complexes with phytochelatins). It can therefore be
speculated that the reduction of As(V) to As(III) plays
an important role in the detoxification of As in higher
plants.
It has been argued that phytochelatins are involved
in differential (inducible) metal tolerance, i.e. natu-
rally or artificially selected intraspecific heritable dif-
ferences in the ability to tolerate exposure to high
levels of metals [58–61]. However, many comparisons
between tolerant and non-tolerant populations have
M. Quaghebeur and Z. Rengel
been made either after a long-term exposure (weeks)
or at highly toxic levels of metal exposure. Therefore,
the evidence in favour of a role for phytochelatins in
differential metal tolerance is often not convincing
[62, 63]. Indeed, further research concluded that dif-
ferential tolerance mechanisms for Cd [63] and Cu
[62] in Silene vulgaris were not due to differential
production of phytochelatins. Similarly, there is evi-
dence that the production of phytochelatins upon
exposure to As(V) is just a part of normal constitutive
As(V) tolerance [40]. However, it has also been
concluded that phytochelatins play a role in As(V)
hypertolerance because higher concentrations of phy-
tochelatins were accumulated in As(V)-tolerant phe-
notypes of Holcus lanatus, and the As(V) tolerance
was completely removed by the presence of the inhib-
itor of glutathione synthesis (buthionine sulfoximine)
[55, 64].
Measurement of Arsenic Speciation
in Terrestrial Plant Material
Arsenic speciation in biological material is generally
determined using ex situ techniques, which are ac-
complished in three steps: sample preparation, species
separation and detection [65]. Given that As species
may be converted from one form to another, or lost
from the sample [46], the most critical step in the
process is undoubtedly sample preparation. It is
necessary to extract quantitatively the As species from
the complex biological matrix while maintaining the
species integrity.
Sample Extraction for Ex-Situ As Speciation
Most of the methods to extract As species from
biological material are optimised to extract organic
As species from marine organisms [66–68]. Polar
organic solvents are often used, but they are not effi-
cient in extracting inorganic As species [69, 70]. More
recent methods focused on the extraction of As spe-
cies from plant tissues by using accelerated solvent
extraction, microwave extraction or enzymatic diges-
tion followed by sonication [71–76] (Table 1). In
some of these methods the extraction efficiencies
were still low [72, 73] or the sum of the inorganic
As species was reported rather than the concentra-
tion of individual inorganic As species [71]. However,
in most recent methods clear analytical distinction
was achieved even between different inorganic As
Table 1. Extraction techniques for As species in terrestrial plant material
Extraction solvent Extraction method Sample Extraction References
efficiency (%)
Methanol-water (9:1) sonication Fungi (Sarcosphaera coronaria, 90  12% [86]
Laccaria amethystina, Sarcodon
imbricatum, Entoloma lividum,
Agaricus haemorrhoidarius, Agaricus
placomyces, Lycoperdon perlatum)
Methanol-water (1:9) microwave-assisted extraction Carrot (Daucus carota) 46 to 69% [72]
Water, methanol-water (1:9) pressurised liquid extraction Holcus lanatus 58 to 94% [89]
Water centrifugation Mung bean (Vigna radiata) 94 to 101% [86]
Water or methanol-water (9:1) shaking Lichens (Alectoria ochroleuca, Usnea water, 7 to 71% [89]
articulata) and higher green plants methanol-water,
(Achillea millefolium, Alnus incana, 4 to 22%
Asplenium viride, Dryopteris dilata,
Equisetum pratense, Fragaria vesca,
Rubus idaeus, Vaccinium myrtilus,
Vaccinium vitis idaea, Picea abies,
Larix deciduus and Deschampsia
caespitosa)
Methanol-water with or sonication Apple (Malus sp.) 79 to 117% [71]
without amylase treatment
As Speciation Governs Arsenic Uptake and Transport in Terrestrial Plants

Trifluoroacetic acid (TFA) incubation Rice (Oryza sativa) 92 to 102% [90]

Methanol-water (1:1) sonication and digestion Rice methanol-water, [19]
trifluoroacetic acid (TFA) 2 M at 100  C 10 to 20% TFA,
105  14%
Water accelerated solvent extraction Carrot 80 to 102% [73]
Water sonication Fern (Pityrogramma calomelanos) 67  24% [91]
Methanol-water (1:1) ultrasonic treatment Fern (Pteris vittata) 60 to 100% [92]
Water, methanol-water (1:1, 9:1) heating in oven at 55  C and Rice water: 93% [93]
ultrasonic treatment methanol-water:
(3 extraction cycles) 86 to 96%
Sucrose (0.33 M), MES (50 mM), microwave-assisted extraction Brasica napus, Holcus lanatus, 104  12% [74]
EDTA (5 mM) and L-ascorbate Bromus rubens, Arabidopsis
(5 mM), pH 5.5 thaliana, Senna planitiicola
Water accelerated solvent extraction Silene vulgaris 73 to 98% [75]
25 mM ammonium acetate ultracentrifugation (30 min at Brassica juncea 47 to 75% [78]
(pH 4.4; 5.6 and 7.8) 10,000 g, 4  C)
Water-ethanol (1:1) microwave-assisted extraction Rice straw (L.) 90% [76]
145
146
species, with high and stable extraction efficiencies
[74–76]. This is important because differentiating bet-
ween As(V) and As(III) in plant material is essential
for understanding how As is metabolised in higher
plants.
In most studies measuring ex situ As speciation in
plant material [71–75], As species were extracted
from plant material without further investigation of
the chemical coordination of different As species.
Raab et al. [77] investigated the stability of synthe-
sized As biomolecules (As-glutathione complexes)
that were spiked to plant material during extraction
procedures. They found that the stability of the As-
glutathione complexes depended on the nature of the
complexes and the type of the extracting solution
used. Reasonable recoveries were obtained for most
As-glutathione complexes when water-methanol or
water-acetonitrile was used as extracting solutions.
In addition, disintegration of As-glutathione com-
plexes is likely to be swift at room temperature
(25  C). Low temperature (4  C) and acidic buffering
solutions (pH 2.5) guaranteed the longest stability
[77].
Separation and Determination Techniques
for Ex-Situ As Speciation
The most commonly used technique for separation of
As species is chromatography, mostly high-perfor-
mance liquid chromatography (HPLC), whereas gas
chromatography (GC) and capillary electrophoresis
(CE) are used to a lesser extent [78]. For As speciation
in plant extracts, ion exchange is the main form of
HPLC separation (Table 2), although ion-pairing and
size exclusion chromatography (SEC) can be used as
well [78]. Even though ion exchange can be used to
separate various organic and inorganic As species
(Table 2), it cannot be used to determine As bio-
molecules [77, 79]. Size exclusion chromatography
(SEC) is most widely used for determination of
metallo-complexes in plant extracts [79]. However,
Raab et al. [77] found only slight separation of differ-
ent As-glutathione (As-GS) complexes [AsIII(GS)3,
MAIII(GS)2, DMAIII(GS)] using this method. So far,
reversed phase chromatography (RFC), with an aceto-
nitrile 0.1% (v=v) formic acid gradient as the
mobile phase, allows the best separation and detection
of As-glutathione complexes, although disintegration
of the As-glutathione complexes was observed (up to
50%) [77].
M. Quaghebeur and Z. Rengel
The speciation of trace levels of As in environmen-
tal and biological samples requires high-sensitivity
detection [78]. In the 1980s, flame atomic absorption
spectrometry (FAAS) was often used for As quantifi-
cation after HPLC separation. However, FAAS has
low sensitivity and high background noise; hence,
the use of this technique declined over the years.
Instead, most recent applications of AAS are com-
bined with hydride generation (HG) [78, 80, 81].
Inductively coupled plasma atomic emission spectro-
metry (ICP-AES) can also be linked to HPLC for
determination of As species. The technique, however,
is only suitable for samples containing high levels of
As [78].
The coupling of HPLC with ICP-Mass Spectrome-
try (ICP-MS) is now the most common technique for
determination of As speciation in environmental sam-
ples (Table 2). The HPLC-ICP-MS technique has high
sensitivity, the multi-element capability and the wide
concentration range for As species [78]. Moreover, it
has recently been shown that double-focusing sector
field ICP-MS (ICP-SFMS) can be used to determine
As species with even higher sensitivity than that of
traditional quadrupole ICP-MS [82]. Hence, HPLC-
ICP-SFMS can be used to determine ultra-trace levels
of As species in the environment.
Despite the high sensitivity, one of the disadvan-
tages of HPLC-ICP-MS is that only an element-spe-
cific signal is generated. Hence, the detection is based
on matching retention times with standards [83]; a
limited number of commercially-available arsenic
standards make the reliability of this method of iden-
tification somewhat questionable [83]. Given the vari-
ety of arsenic compounds in the environment, it is
often necessary to collect the fractions with unknown
peaks or co-eluted As-containing peaks for further
chromatographic separation (multidimensional chro-
matography), or for structural identification with elec-
trospray mass spectrometry (ES-MS) [82]. ES-MS is a
mode of analysis providing molecular and structural
information that has been used for characterising
arsenosugars in macro-algae and determination of
As species in marine fauna [83]. ES-MS has also been
used to study As-containing biomolecules (e.g. As-
phytochelatin complexes) in terrestrial plants [54,
77, 78, 84]. These complexes have also been deter-
mined with an electrospray-quadrupole-time of flight
(ESI-Q-TOF) detection system (e.g. in Brassica
juncea=Indian mustard=[79]). A comprehensive re-
view article about available methods for determina-
Table 2. Separation of As species in terrestrial plant material using chromatography
Column Mobile phase Flow rate Detection Arsenic species References
(mL min1 )
Hamilton PRP-X100 30 mM NaH2PO4=Na2HPO4, pH 6 1.5 ICP-MS As(III), As(V) [86]
Anion exchange
Hamilton PRP-X100 20 mM NH4H2PO4, pH 5.6 ICP-MS As(III), As(V), DMA, MMA [89]
Anion exchange
Hamilton PRP-X100 10 mM NH4H2PO4, 10 mM NH4NO3, pH 6.2 1.0 ICP-MS As(III), As(V), DMA, MMA [71, 90]
Anion exchange
Hamilton PRP-X100 30 mM H3PO4, pH 6.0 1.0 ICP-MS As(III), As(V), DMA, MMA [19, 74]
Anion exchange
Hamilton PRP-X100 20 mM NH4H2PO4, pH 5.6 1.5 ICP-MS As(III), As(V), DMA, MMA [74, 91]
Anion exchange
Hamilton PRP-X100 0.015 M K2HPO4, 0.015 M KH2PO4, pH 5.9 1.0 ICP-MS As(III), As(V), DMA, MMA [92]
Anion exchange
Hamilton PRP-X100 10 mM (NH4)2CO3, pH 8.0 ICP-MS As(III), As(V), DMA, MMA [77]
Anion exchange
Hamilton PRP-X100 10 mM citric acid, pH 2.0 ICP-MS As(III), As(V), DMA, MMA [77]
Anion exchange
Supercosil LC-SAX 30 mM NaH2PO4, pH 3.75 1.5 ICP-MS As(III), As(V), DMA, MMA, [86]
Anion exchange AsB, TMAO
As Speciation Governs Arsenic Uptake and Transport in Terrestrial Plants

IC-PAK AHR 10 mM NaH2PO4 (80%), 10 mM 0.5 ICP-MS As(III), As(V), DMA, MMA, AsB [88]
Anion exchange Na2HPO4 (20%), pH 6.0
IC-Pak Anion HR 10 mM (NH4)2CO3, pH 10.0 1.0 ICP-MS As(III), As(V), DMA, MMA, AsB [73, 90]
Anion exchange
IonPac AS7=AG7 0.4 to 50 mM HNO3 (gradient) with 0.9 ICP-MS As(III), As(V), DMA, MMA, [89]
Anion exchange 0.05 mM benzene-1,2-disulfonic acid AsB, AsC, TMAO, TMA
IonPac AS7=AG7 12.5 mM HNO3, pH 1.8 0.5 ICP-MS As(III), As(V), DMA, MMA, AsB, [90]
Anion exchange AsC, TMA, TMAO
IonPac AS7=AG7 50 mM HNO3 (gradient) with 0.4 mM HNO3 ICP-MS As(III), As(V), DMA, MMA, AsB, [75]
Anion exchange AsC, TMA, TMAO
ION-120 45 mM (NH4)2CO3 in water-methanol 1.0 ICP-MS As(III), As(V), DMA, mMA [72]
Anion exchange (97:3), pH 10.3
IonPac AS11 100 mM NaOH 1.0 HG-AFS As(III), As(V), DMA, MMA [76]
Zorbax 300-SCX 20 mM pyridine, pH 2.6 ICP-MS AsB, AsC, TMAO, TETRA [89, 91]
Cation exchange
Supercosil LC-SCX 20 mM pyridine, pH 2.6 1.5 ICP-MS As(III), AsC, AsB [74, 77]
Cation exchange

(continued)
147
148 M. Quaghebeur and Z. Rengel

References
tion of arsenic species in different matrices has
recently been published by Francesconi and Kuehnelt

[107]
[93]

[77]

[78]
[85].

In-situ As Speciation

As(III), DMA, mMA, As(V)

In contrast to the ex situ techniques that require the
AsIII(GS)3, MAIII(GS)2,

extraction of As species from the plant matrix before

separation by chromatography and determination of
Arsenic species

the As species in the extracts by spectrometric detec-

As(III), As-PC
As(III), AsB

DMAIII(GS)

tion techniques, the in situ X-ray absorption spectro-

scopy (XAS) technique allows determination of As
species directly in plant material with minimal sample
preparation [53, 95, 96]. Hence, XAS decreases the
possibility of spurious changes in As speciation dur-
ing extraction and sample preparation. In addition,
ICP-MS=ESI-MS

XAS can be used to determine the local coordination

environment of As in diverse plant species and tissues
ESI-Q-TOF

[53, 96]. However, the major disadvantages of the

Detection

ICP-MS=
ICP-MS

ICP-MS

XAS technique are (i) the detection limit for As spe-

cies is rather high (between 2 and 10 mg As kg1 ,
depending on the quality of the X-ray beam), and
(ii) the technique only detects the major species of
As present in the plant. It is, therefore, likely that
(mL min1 )

As species present in low concentrations in plant tis-

Flow rate

sues (i.e. representing <5% of the total As) are not

1.5

detected [96]. In contrast, the detection limits of the

ex situ techniques are very low (<1 mg As kg1 ); these
techniques also allow independent determination of
different As species.
Gradient: 0 to 20 min up to 5 to 30% B,

25 mM ammonium acetate (pH 5.6; 7.8)

Eluent A: 0.1% formic acid in water

Arsenic Speciation and Regulation

4 mM pyridine=formiate, pH 2.8

TMAO Trimethylarsine oxide; TETRA tetramethylarsoinum ion.

bromide in 20 mM ammonium
hexadecyltrimethylammonium

in Higher Plants
2% (v=v) methanol, pH 6.0

Phytoplankton, macroalgae and fungi contain large

Eluent B: acetonitrile

amounts of organic As species [86, 97], whereas ter-

98% (v=v) 10 mM
then 10 min 5% B

restrial plants contain mainly inorganic As [As(III)

phosphate buffer
Mobile phase

and As(V)] [88, 91, 98, 99]. Small amounts of organic

As species have also been detected in plant tissues,
but it is unclear whether these organic As species are
produced in plant metabolism from inorganic As
taken up, or whether they are actually taken up from
soil as organic As species [20].
Arsenic species (both organic and inorganic) are
TSK Gel-2000 GSXW

toxic to plants. Early research showed that rice grown

Hamilton PRP-X200
Table 2 (continued)

Waters ODS2 C18

in soil treated repeatedly with MMA showed symp-

Cation exchange

Haisil 100 C18

Reverse phase

Reverse phase

Size exclusion

toms of straight-head disease [100]. The inorganic

As species (arsenate and arsenite) cause a variety of
Column

symptoms ranging from inhibition of root growth to

plant death [101]. Arsenite is a well-known thiol
As Speciation Governs Arsenic Uptake and Transport in Terrestrial Plants
reagent that combines rapidly with dithiol groups on
proteins and is hence an effective inhibitor of enzymes
requiring free sulfhydryl groups [102]. Arsenate, on
the other hand, is a competitive inhibitor of phosphate
[103] and acts as an uncoupler of oxidative phosphor-
ylation [102]. Arsenate competes with phosphate for
binding to ADP; the formation of the As(V)-ATP ana-
logues deprives the cells of energy source, ultimately
leading to cell death [23].
Reactive oxygen species (ROS) are generated upon
exposure to inorganic arsenic species, resulting in sig-
nificant lipid peroxidation [104]. This probably occurs
through the conversion of arsenate to arsenite, a pro-
cess which readily occurs in plants and leads to the
synthesis of enzymatic and non-enzymatic antioxi-
dants [104].
In general, it can be stated that inorganic arsenicals
are more toxic than organic arsenicals and that the
trivalent oxidation state is more toxic than the penta-
valent oxidation state [1, 2]. However, for terrestrial
plants, varied results have been reported. In a study
with Spartina patens investigating the phytotoxicity
of four arsenic species, the order of phytotoxicity
was reported as As(V) ffi As(III)<MMA<DMA,
suggesting that organic arsenicals were more toxic
than inorganic arsenic species [30]. For rice grown in
hydroponic culture, however, the order of phytotox-
icity was DMA<As(V)<MMA<As(III), suggesting
that for terrestrial plants the degree of phytotoxicity of
different arsenicals can vary according to the plant
species and environmental conditions considered.
Arsenicals have been shown to bind to a variety of
cytosolic proteins and macromolecular constituents of
tissues [105]. Arsenite is likely to be bound strongly
to –SH groups of cytosolic proteins and macromole-
cular constituents [105]. The affinity of arsenate for
–SH groups is however much lower [57].
Recent studies have described the formation of
As(III)-phytochelatin (As-PC) complexes in several
terrestrial plants upon exposure to As(V) [52–55].
Hence, it is likely that plants are able to reduce
As(V) to As(III), with As(III) readily forming com-
plexes with thiol groups in phytochelatins. The As-PC
complexes are stable at acidic pH, but dissociate into
phytochelatin and free As(III), with the latter getting
oxidised to As(V) above pH 7.5 [57]. Therefore, it is
generally assumed that As(III) is the dominant As
species in terrestrial plants, regardless of plant part
(roots or shoots), plant species or the As(V) and
P(V) concentrations in the growth medium. The pre-
149
sence of As(V) in plant extracts has, hence, often been
regarded as an artifact of the extraction procedure
because As(V) may have originated from phytochela-
tin-complexed As(III) [20]. Both As(V) and As(III)
were, however, measured in shoots of Pteris vittata
using x-ray absorption near edge structure spectro-
scopy (XANES) [95]. Given that this technique allows
determination of in situ As speciation in plant material
without the need for extraction, it is unlikely that
As(V) reported in Pteris vittata (25% of total As)
would be an artifact of sample preparation.
It is clear that plants can reduce As(V) to As(III)
because both ions have been reported in tissues of
plants supplied with As(V) [20, 53, 88]. In vitro, this
reduction is facilitated non-enzymatically by glu-
tathione [106], which may be present at high cellular
concentrations in plant tissue [25]. However, nothing
is known about the mechanisms of the reduction of
arsenate in plants [75].
Regulation of As speciation in terrestrial plants
is poorly understood. Nevertheless, As speciation in
Holcus lanatus was recently characterised by studying
the occurrence, distribution and dynamics of arsenic
species [74]. Evidence was provided that (i) As spe-
ciation is closely regulated by As(V) influx, and (ii)
that As speciation plays an important role in As detox-
ification in Holcus lanatus. At low As(V) influx,
As(V) was partially reduced to As(III), but the pro-
portion of As(V) reduced to As(III) increased with
increasing As(V) influx. In addition, the linear corre-
lation was found between As(III) concentration in
roots and the concentration of total As in roots [74].
Therefore, plants protect roots by reducing As(V) to
As(III), with As(III) most likely being detoxified by
complexation to phytochelatins and transported out of
the cytoplasm into the vacuole. An interesting obser-
vation is that the absence of either As(V)-specific
internal detoxification or an efflux system is wide-
spread in nature (bacteria, yeast, terrestrial plants
and mammals) [107].
Current studies elucidating the occurrence and reg-
ulation of As speciation in plant tissues have mostly
measured As species by ex situ techniques. However,
the extraction of As species and the traditional chro-
matographic separation may result in the disintegra-
tion of As(III)-glutathione complexes [77]. Therefore,
using ex situ As speciation does not provide unequiv-
ocal answers to whether the As(III) measured in plant
extracts represents free As(III) in plant tissue, or whether
it is the result of dissociation of As(III)-phytochelatin
150
complexes during preparation of plant extracts.
Moreover, further research is required to characterise
whether dissociation of As(III)-phytochelatin com-
plexes results in free As(III) and phytochelatins, or
whether further oxidation of As(III) to As(V) takes
place during ex situ As speciation.
Arsenic Translocation and Distribution
in Higher Plants
Different plant species accumulate As in different
parts, with mobility or translocation of As in a plant
being influenced by the form of As available [21]. The
organic As species (MMA and DMA) are generally
poorly translocated from roots to shoots [17, 30, 108],
whereas the translocation of the inorganic Arsenic spe-
cies varies greatly between plant species and is influ-
enced by As(V) and P(V) supply. Because of the
chemical similarity between P(V) and As(V), it might
be expected that As(V) is translocated via the same
system as P(V). In Holcus lanatus, the translocation
of As from roots to shoots indeed increased with
increasing P(V) concentration in the nutrient solution
[26]. However, in Urtica dioica the As concentration in
various tissues did not correlate with P concentration in
these tissues under varying P(V) supplies, suggesting
that As may not be translocated via the P(V) pathway
[109]. It is worth noting that in both Holcus lanatus and
Urtica dioca the relation between P and As transloca-
tion was studied without considering As speciation in
plant material. On the other hand, both As(III) and
As(V) were found in the xylem sap of Brassica juncea,
but it remained unclear whether both As(III) and As(V)
were loaded as such in the xylem sap, or whether trans-
formation between As species did occur in the xylem
sap. In contrast, As(III) appeared to be the main As
species loaded in the xylem of the hyperaccumulating
fern Pteris vittata [110, 111]. This assumption was
based on (i) the concentrations of P in both roots and
shoots of different Pteris species not being significantly
affected by the addition of As(V) to the substrate, and
(ii) no hyperaccumulation of P occurring in the fern.
However, no direct evidence is available about the form
of As translocated from roots to shoots, with most
reports not including measurements of As speciation
in roots of hyperaccumulating ferns (Pteris vittata
[95, 96], Pteris cretica, Pteris longifola and Pteris
umbrosa [110]).
In recent studies As(V) uptake and speciation was
studied in two mutants of Arabidopsis thaliana L.
M. Quaghebeur and Z. Rengel
[pho1 (P deficient) and pho2 (P accumulator)] with
defects in the regulation of uptake and translocation
of P(V) from roots to shoots [112]. It can be postu-
lated that the translocation of As(V) from roots would
occur via the same pathway as P, whereas the trans-
location of As(III), if any, would occur via a different
pathway. The results [112] indicated that P and As
were not translocated via the same pathway in the
pho1 and pho2 mutants. Hence, it is likely that As(III)
rather than As(V) is the predominant As species
loaded in the xylem in Arabidopsis thaliana.
Most plants supplied with As(V) in the root medi-
um reduce a large proportion of As(V) taken up to
As(III) in roots (57 to 100%), with As(III) most likely
then being complexed to phytochelatins. Hence, these
plants use the reduction of As(V) to As(III) as the first
step in detoxifying As (as discussed above) and
therefore contain mainly As(III) in roots [53, 75, 88,
112, 113]. These plants generally translocate only a
small proportion of the total As from roots to shoots.
In contrast, As-hyperaccumulating plants contain
mostly As(V) in roots [>90% of total As] [91, 92,
114], and translocate to shoots most of As taken up
(50 to 78%) [92, 111]. Hence, it is likely that the As-
hyperaccumulating plants do not use the reduction of
As(V) to As(III) and subsequent complexation of
As(III) to phytochelatins in roots as the As-detoxifica-
tion strategy in contrast to other plant species.
Therefore, it could be speculated that the intense
translocation of As from roots to shoots (e.g. in As-
hyperaccumulating ferns) is a way of protecting roots
from As toxicity. This would imply that As-hyperac-
cumulating ferns do not have an efficient mechanism
of complexing As(III) to phytochelatins. It has indeed
been suggested that the complexation of As(III) to
phytochelatins is unlikely to play a significant role
in the detoxification of As in shoots of Pteris vittata
[110], but the unequivocal evidence has yet to be
reported.
Conclusions
Regulation of As speciation in terrestrial plants is
poorly understood albeit it is essential for charac-
terising cycling of As in the soil-water-plant-human
continuum. Arsenic species need to be extracted
quantitatively (and without loss of species integrity)
from plant material before being quantified by HPLC-
ICP-MS. Arsenic speciation may be regulated by As
influx. Plants have a capacity to reduce As(V) to
As Speciation Governs Arsenic Uptake and Transport in Terrestrial Plants
As(III) which is then complexed with phytochelatins or
loaded into the xylem sap for translocation to shoots.
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