Selenium Assimilation and Volatilization from
Selenocyanate-Treated Indian Mustard and Muskgrass1
Mark P. de Souza, Ingrid J. Pickering, Michael Walla, and Norman Terry*
Department of Plant and Microbial Biology, University of California, Berkeley, California 94720–3102
(M.P.d.S., N.T.); Stanford Synchrotron Radiation Laboratory, Stanford Linear Accelerator Center, Menlo
Park, California 94025–7015 (I.J.P.); and Department of Chemistry and Biochemistry, University of South
Carolina, Columbia, South Carolina 29208 (M.W.)
Selenocyanate (SeCN⫺) is a major contaminant in the effluents from some oil refineries, power plants, and in mine drainage
water. In this study, we determined the potential of Indian mustard (Brassica juncea) and muskgrass (a macroalga, Chara
canescens) for SeCN⫺ phytoremediation in upland and wetland situations, respectively. The tolerance of Indian mustard to
toxic levels of SeCN⫺ was similar to or higher than other toxic forms of Se. Indian mustard treated with 20 ␮m SeCN⫺
removed 30% (w/v) of the Se supplied in 5 d, accumulating 554 and 86 ␮g of Se g⫺1 dry weight in roots and shoots,
respectively. Under similar conditions, muskgrass removed approximately 9% (w/v) of the Se supplied as SeCN⫺ and
accumulated 27 ␮g of Se g⫺1 dry weight. A biochemical pathway for SeCN⫺ degradation was proposed for Indian mustard.
Indian mustard and muskgrass efficiently degraded SeCN⫺ as none of the Se accumulated by either organism remained in
this form. Indian mustard accumulated predominantly organic Se, whereas muskgrass contained Se mainly as selenite and
organic Se forms. Indian mustard produced volatile Se from SeCN⫺ in the form of less toxic dimethylselenide. Se
volatilization by Indian mustard accounted for only 0.7% (w/v) of the SeCN⫺ removed, likely because the biochemical steps
in the production of dimethylselenide from organic Se were rate limiting. Indian mustard is promising for the phytoremediation of SeCN⫺-contaminated soil and water because of its remarkable abilities to phytoextract SeCN⫺ and degrade all the
accumulated SeCN⫺ to other Se forms.
Selenocyanate (SeCN⫺) is a major pollutant in effluents from some oil refineries and power plants,
and especially in mining wastewater when cyanide
leaches selenide (Se2⫺) ores. Physicochemical methods for the removal of SeCN⫺ have been investigated; these methods include ion exchange and precipitation with Cu, Ag, Au, Cd, Hg, Th, and Pb
(Manceau and Gallup, 1997). These methods are, unfortunately, very expensive, often requiring large and
toxic amounts of heavy metals to precipitate SeCN⫺.
A more cost-effective method for the clean up of
large volumes of SeCN⫺ may be the use of constructed wetlands, as was shown for oil refinery effluent contaminated with selenite (SeO32⫺; Hansen et
al., 1998). Vegetated flow-through wetland microcosms successfully cleaned up SeCN⫺-contaminated
wastewater from a power plant, removing 79% and
1
This work was supported by the Cinergy Corporation and by
the Electric Power Research Institute (grant nos. W08021–30 and
W04163). The XAS analysis was performed at the Stanford Synchrotron Radiation Laboratory, which is funded by the U.S. Department of Energy, Offices of Basic Energy Sciences and Biological and Environmental Research, by the National Institutes of
Health, National Center for Research Resources, Biomedical Technology Program, and by the National Institute of General Medical
Sciences.
* Corresponding author; e-mail [email protected]; fax
510 – 642– 4995.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.010686.
54% (w/v) of the mass of Se and CN⫺, respectively,
from the inflow (S.N. Whiting and N. Terry, unpublished data). However, before vegetated wetlands are
used for the remediation of SeCN⫺-contaminated
water, it is essential to determine the fate of the two
toxic components of SeCN⫺, i.e. Se and cyanate
(OCN⫺), in plant tissues and sediments. X-ray absorption spectroscopy (XAS) is an excellent analytical
tool for identifying different forms of Se in vivo. In
the current study, we have used XAS to determine
the fate of SeCN⫺ in Indian mustard (Brassica juncea)
and muskgrass (a macroalga, Chara canescens). These
two species were selected because they are excellent
candidates for the phytoremediation of many different trace elements, including Se, and are potentially
important for phytoremediation in two different situations, uplands and wetlands.
Indian mustard is useful for the phytoremediation
of contaminated upland soil via phytoextraction, the
accumulation of contaminants in biomass (Kumar et
al., 1995). Experiments with Se-contaminated agricultural soil have shown that Indian mustard is one of
the best plants tested so far for Se phytoremediation,
with almost 50% (w/v) of the soil Se removed by the
plants in three crops (Bañuelos and Meek, 1990; Bañuelos et al., 1995). Indian mustard was also effective
in removing contaminants from water in a process
known as rhizofiltration (Dushkenov et al., 1995). In
addition to being an excellent candidate for the phytoextraction of Se from soil and water, Indian mus-
Plant Physiology, February 2002, Vol.
Downloaded
128, pp. 625–633,
from on June
www.plantphysiol.org
18, 2017 - Published
© by
2002
www.plantphysiol.org
American Society of Plant Biologists
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
625
de Souza et al.
tard is also promising for phytovolatilization, i.e. the
production of volatile Se from inorganic or organic Se
present in contaminated soil and water (Terry et al.,
2000). Phytovolatilization has the advantage of removing Se from the site in a relatively nontoxic form
(Lin et al., 2000). Dimethylselenide (DMSe), the major
volatile form of Se produced by most nonhyperaccumulator plants like Indian mustard, is 500 to 700
times less toxic than selenate or selenite (Wilber,
1980).
Chara sp. are macroalgae that make excellent candidates for metal(loid) phytoremediation because
they produce a large biomass under field conditions
(Carneiro et al., 1994; Herrera-Silveira, 1994) and
bioconcentrate large amounts of trace elements (Ye et
al., 2001). Muskgrass in the Allegheny Power Service
wetland at Springdale, Pennsylvania, accumulated
concentrations of iron and manganese from coal ash
leachate that were orders of magnitude higher than
vascular plants such as cattail growing in the same
wetland (Ye et al., 2001). Muskgrass has also been
suggested as a candidate for the remediation of
selenate at the Kesterson Reservoir in the San Joaquin
Valley, California (Horne, 2000).
The two major objectives of the research described
here are to determine whether Indian mustard and
muskgrass may be used for the phytoremediation of
SeCN⫺ and to determine the metabolic fate of SeCN⫺
in plant tissues. To achieve the first objective, the
tolerance of Indian mustard and muskgrass to toxic
levels of SeCN⫺ was determined, along with their
abilities to phytoextract and phytovolatilize Se when
supplied with SeCN⫺. In this regard, Indian mustard
and muskgrass were treated with SeCN⫺ and their
biomass production and rates of Se accumulation and
volatilization were measured under controlled conditions. With regard to the second objective, XAS was
used to determine the fate of SeCN⫺ accumulated in
tissues. These data were used to propose a model for
SeCN⫺ assimilation in Indian mustard as a model
plant.
RESULTS
For plants to be used for SeCN⫺ phytoremediation,
they must be able to tolerate high levels (200 ␮m or 16
mg L⫺1) of Se similar to or higher than the levels
found in SeCN⫺-contaminated sites. The wastewater
from the sour water stripper of a coal gasification
plant in Indiana contained SeCN⫺ at 1.4 mg L⫺1 (S.N.
Whiting and N. Terry, unpublished data). When the
different Se forms were supplied at 200 ␮m, Indian
mustard seedlings were more tolerant of SeCN⫺ and
selenate than selenite, as they had significantly
higher fresh weights as compared with those grown
with 200 ␮m selenite (Fig. 1; P ⬍ 0.05). Although the
root lengths of plants grown with 200 ␮m SeCN⫺
were not significantly different from those grown in
the presence of 200 ␮m selenate or selenite (P ⬎ 0.05),
626
Figure 1. Tolerance of Indian mustard seedlings to selenate, selenite,
and SeCN⫺. Seedlings were germinated in one-half-strength Murashige and Skoog agar containing the different chemical forms of Se at
concentrations of 0, 20, or 200 ␮M. Vertical bars indicate 1 SE from
the mean, n ⫽ 24. Bars showing the same letter code within each
graph are not significantly different (P ⬎ 0.05).
inferences cannot be made regarding Se tolerance
because seedlings grown with 200 or 20 ␮m SeCN⫺
had multiple short roots. Seedlings grown under control conditions or those grown with 20 or 200 ␮m
selenate or selenite had single roots. Seedlings in the
200 ␮m selenate treatment had significantly longer
roots than those in the 200 ␮m selenite treatment (P ⬍
0.05), suggesting that Indian mustard is more tolerant of toxic levels of selenate than selenite. At 20 ␮m
Se, the fresh weights of Indian mustard grown in the
presence of selenate, selenite, or SeCN⫺ were similar
to each other, but the root lengths of the SeCN⫺
treatment were significantly shorter than those in the
selenate or selenite treatments because they had multiple roots.
Indian mustard seedlings rapidly accumulated Se
and cyanide in their tissues when supplied with 100
␮m SeCN⫺ (197 ␮g of Se and 32.5 ␮g of cyanide). In
0.5 h, the seedlings accumulated 15 ⫾ 3 ␮g Se and
9.9 ⫾ 1.7 ␮g cyanide. Since plants were shown to take
up SeCN⫺, the next experiments were designed to
determine the capacity of Indian mustard and
muskgrass for the phytoextraction and phytovolatilization of Se when they were supplied with SeCN⫺.
Indian mustard showed a remarkable ability to
accumulate Se in its tissues when the plants were
supplied with 20 ␮m SeCN⫺ in hydroponic solution.
The root Se concentrations reached 554 ␮g g⫺1 dry
weight after treatment for 5 d with SeCN⫺ in hydro-
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 128, 2002
Se Assimilation and Volatilization from Selenocyanate
ponic solution (Fig. 2). The shoots (harvestable biomass) of the SeCN⫺-supplied plants accumulated
lower concentrations of Se (86 ␮g g⫺1 dry weight) as
compared with the shoots or roots of the selenatesupplied plants and the roots of the selenite-supplied
plants (124–177 ␮g of Se g⫺1 dry weight). The accumulation of Se from SeCN⫺ did not involve a bacterial role as was shown earlier for selenate accumulation in Indian mustard (de Souza et al., 1999). Axenic
Indian mustard seedlings accumulated 190 ⫾ 22 ␮g
g⫺1 dry weight of tissue (root ⫹ shoot) when they
were grown for a 10 d period on agar containing 20
␮m SeCN⫺. When axenic seeds were coated with a
mixture of rhizosphere bacteria and germinated on
agar containing SeCN⫺, the seedlings contained
182 ⫾ 17 ␮g Se g⫺1 dry weight, a concentration that
was not significantly different (P ⬎ 0.05) from the
similarly treated axenic seedlings.
Unlike Indian mustard where only the shoots can
be easily harvested, all the tissue of muskgrass is
harvestable biomass because this macroalga is free
living or loosely attached to the substrate. Unfortu-
Figure 3. Se volatilization by Indian mustard (A) and muskgrass (B)
supplied with selenate, selenite, or SeCN⫺. All Se species were
supplied at 20 ␮M in hydroponic solution. Vertical bars indicate 1 SE
from the mean, n ⫽ 4. The rates of Se volatilization and the statistical
differences between the different lines are presented in Table I. The
dry weights of the plants are shown in the legend to Figure 2.
Figure 2. Se concentrations in tissues of Indian mustard and
muskgrass supplied with selenate (A), selenite (B), or SeCN⫺ (C). All
Se species were supplied at 20 ␮M in hydroponic solution. Vertical
bars indicate 1 SE from the mean, n ⫽ 4. Bars showing the same letter
code in all parts of the figure are not significantly different from each
other (P ⬎ 0.05). The biomass (fresh weight) of the plants did not
change over the 5-d exposure to Se. The dry weights at the end of the
5-d period for selenate, selenite, and SeCN⫺-supplied Indian mustard
were 0.58 ⫾ 0.21, 0.62 ⫾ 0.14, and 0.60 ⫾ 0.10, respectively, and
for muskgrass were 0.40 ⫾ 0.028, 0.37 ⫾ 0.028, 0.40 ⫾ 0.046,
respectively.
Plant Physiol. Vol. 128, 2002
nately, muskgrass accumulated relatively lower Se
concentrations in its tissues when supplied with
SeCN⫺, selenate, or selenite (11, 7, and 30 ␮g g⫺1 dry
weight), compared with Indian mustard (Fig. 2).
Indian mustard and muskgrass treated with 20 ␮m
SeCN⫺ in hydroponic solution were able to produce
volatile Se from SeCN⫺ (Fig. 3). The volatile Se species produced by Indian mustard treated with
SeCN⫺, selenate, or selenite was identified by gas
chromatography/mass spectrometry (GC/MS) as
DMSe. No dimethyldiselenide (DMDSe) or hydrogen
selenide (H2Se) was detected. The production of
CH3SeCN cannot be ruled out because its mass is
similar to DMSe and it may have had a similar retention time to DMSe on the GC column. Therefore,
the rates of Se volatilization by Indian mustard and
muskgrass were measured by trapping all volatile Se
species produced in an alkaline peroxide solution
using the chamber and trap method described earlier
(Zayed and Terry, 1992). Volatile Se production by
Indian mustard and muskgrass supplied with
SeCN⫺, selenate, or selenite increased linearly with
time (Fig. 3). The rate of Se volatilization by SeCN⫺supplied muskgrass was not significantly different
from that measured from SeCN⫺-treated Indian mus-
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
627
de Souza et al.
tard or from selenate-supplied macroalgae (Table I).
The rate of Se volatilization from SeCN⫺-supplied
Indian mustard was 2-fold higher than the rate obtained when the plant was supplied with selenate
(Table I). Selenite-supplied Indian mustard had a
significantly higher rate of Se volatilization than
SeCN⫺ or selenate-supplied plants (1.3- and 2.8-fold,
respectively). Selenite-supplied muskgrass had 10- to
27-fold higher rates than those measured from the
Indian mustard treatments and 6- and 13-fold higher
than SeCN⫺ or selenate supplied macroalgae,
respectively.
The fate of the accumulated Se in SeCN⫺-supplied
plants was studied by XAS. The spectrum of a reference solution of SeCN⫺ was very different from that
of solutions of selenate or selenite (Fig. 4). The
SeCN⫺ edge was similar to that of Se-Met, but
showed very different post-edge characteristics.
When the XAS spectra of the plant samples were
fitted to the references, it was clear that none of the Se
accumulated in the tissues of Indian mustard or
muskgrass remained in the form of SeCN⫺ (Table II).
In Indian mustard, most of the Se in SeCN⫺-supplied
mature plants showed a spectrum very similar to
organic Se forms such as Se-Met and selenocystine.
Similar results were seen in earlier work where
selenite-supplied Indian mustard accumulated organic forms of Se (de Souza et al., 1998). In contrast,
selenate-supplied Indian mustard plants transformed
very little of the selenate to reduced forms because
ATP sulfurylase, which activates selenate for reduction, is a major rate-limiting step in selenate assimilation (Pilon-Smits et al., 1999). The XAS spectra of Se
in SeCN⫺-supplied Indian mustard seedlings that
were grown axenically or in the presence of bacteria
were similar to each other and similar to the spectra
obtained for mature plants (data not shown). These
XAS data for seedlings indicate that bacteria do not
play a role in SeCN⫺ assimilation by Indian mustard.
Muskgrass transformed SeCN⫺ in a manner very
different from Indian mustard. The Se that had accumulated in the tissues of SeCN⫺-supplied muskgrass
had a spectrum that was fit by equal contributions of
selenite and organic Se. This suggests that muskgrass
does not produce organic Se as efficiently as Indian
mustard. The XAS data from selenite-treated
muskgrass support this hypothesis. When selenite
was supplied, the XAS spectrum of Se accumulated
Figure 4. Se K near-edge x-ray absorption spectra of selenium accumulated by Indian mustard and muskgrass supplied with 20 ␮M
SeCN⫺ (top) compared with the spectra for aqueous solutions of
selenate, selenite, L-selenocystine (seleno-Cys dimer), and Se-Met,
which were used as Se standards.
by muskgrass could be fit with approximately 66%
organic Se and 34% selenite. As mentioned above,
selenite-supplied Indian mustard accumulated
mainly organic Se. Thus, Indian mustard appears to
be more effective at converting SeCN⫺ and selenite to
organic Se than muskgrass. However, muskgrass was
more efficient than Indian mustard at reducing
selenate because a significant proportion (approximately 47%) of the Se accumulated in the tissues of
selenate-supplied muskgrass could be attributed to
Table I. Rates of Se volatilization by Indian mustard and muskgrass
These rates are the slopes of the best-fit lines for the data shown in Figure 3. Also shown are the R2
values for the curve fit and the statistical comparison of the different Se volatilization curves shown in
Figure 3. Values with the same letter code are not significantly different from each other (P ⬎ 0.05).
Treatment
2⫺
4
2⫺
3
⫺
SeO
SeO
SeCN
628
Muskgrass
Indian Mustard
␮g Se g⫺1 dry wt d⫺1
R2
␮g Se g⫺1 dry wt d⫺1
R2
0.53 a
7.11 b
1.16 ad
0.9931
0.9922
0.9769
0.26 c
0.74 d
0.54 a
0.9847
0.9924
0.9978
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 128, 2002
Se Assimilation and Volatilization from Selenocyanate
Table II. Results of fitting Se K near-edge x-ray absorption spectra of Indian mustard and muskgrass
The fractional contribution of standard spectra to the spectrum of the unknown is shown. The values in parentheses are three times the
estimated SD of the last figure(s). R is the least-squares residual, ⌺(Iobs ⫺ Icalc)/N, where Iobs and Icalc are the observed and calculated data, and
N is the number of data points. Elemental Se was also included in the fits, but did not contribute. The selenocystine standard is a stable dimer
of selenocysteine.
SeCN⫺
Treatment
Indian mustard
SeCN⫺
Shoots
Roots
Muskgrass
SeO42⫺
SeO32⫺
SeCN⫺
SeO42⫺
SeMet
Selenocystine
R ⫻ 10
0
0
0
0
0.07 (2)
0.09 (2)
0.75 (6)
0.32 (6)
0.18 (5)
0.59 (5)
0.315
0.329
0
0
0
0.40 (1)
0
0
0.13 (2)
0.34 (1)
0.45 (2)
0.23 (7)
0.39 (5)
0.31 (6)
0.24 (6)
0.27 (4)
0.24 (6)
0.441
0.199
0.351
organic Se, with approximately 40% selenate and
13% selenite. Thus, selenate reduction does not appear to be rate limiting for Se assimilation in
muskgrass, as it appears to be in Indian mustard.
DISCUSSION
Use of Indian Mustard and Muskgrass for the
Phytoremediation of SeCN
The results clearly show that Indian mustard and
muskgrass are suitable for the phytoremediation of
SeCN⫺ in upland and wetland environments, respectively, because they were able to tolerate, take up,
and assimilate SeCN⫺ to organic Se forms and less
toxic DMSe. SeCN⫺ supplied at a relatively high
concentration of 200 ␮m (16 mg L⫺1) was not much
more toxic to Indian mustard than selenate or selenite supplied at the same concentration, suggesting
that plants should be able to tolerate the relatively
lower concentrations of SeCN⫺ encountered in contaminated sites; e.g. the wastewater from the sour
water stripper of a coal gasification plant in Indiana
contained SeCN⫺ at 1.4 mg L⫺1 (S.N. Whiting and N.
Terry, unpublished data).
Indian mustard was much more efficient than
muskgrass at removing Se from the supplied SeCN⫺
in the hydroponic solution. SeCN⫺ was supplied at a
concentration of 20 ␮m (1.6 ␮g of Se mL⫺1). Because
each plant was maintained in 200 mL of hydroponic
solution, 320 ␮g of Se was supplied to each plant. The
mass of Se removed from the hydroponic solution by
Indian mustard and muskgrass was calculated from
the biomass values shown in the legend to Figure 2
and from the tissue Se concentrations and Se volatilization values shown in Figures 2 and 3. Indian mustard roots removed 44 ␮g, shoots removed 50 ␮g, and
Se volatilization removed 0.7 ␮g of the Se supplied as
SeCN⫺. Thus, in 5 d, Indian mustard removed approximately 98 ␮g of Se, i.e. 30% (w/v) of the Se
supplied as SeCN⫺. Muskgrass, however, removed
only 9% (w/v) of the 320 ␮g of SeCN⫺ supplied, with
27 ␮g of Se phytoextracted into its tissue and 2.3 ␮g
of Se volatilized. Muskgrass is better suited for the
phytoremediation of selenite-contaminated water bePlant Physiol. Vol. 128, 2002
SeO32⫺
cause it removed approximately 13% (w/v) of the Se
supplied, approximately 4% and 9% (w/v) by volatilization and phytoextraction, respectively.
Indian mustard was very efficient at accumulating
Se from SeCN⫺-contaminated water because onethird of the Se supplied as SeCN⫺ was accumulated
in its tissue. One-half of the mass of Se removed from
SeCN⫺-contaminated solution was removed by phytoextraction into the shoots. The shoots of Indian
mustard plants used for the phytoremediation of
SeCN⫺-contaminated soil may be harvested to physically remove Se from the site. For Indian mustard to
be used for the phytoremediation of SeCN⫺contaminated water, plants may be grown in a rhizofiltration setup where shoots and roots may be
harvested.
Muskgrass could be used in constructed wetlands
treating SeCN⫺-contaminated water, but its ability to
phytoextract Se over a short period of time was not as
efficient as the upland plant, Indian mustard. There
may be other wetland species more suited for the
phytoremediation of SeCN⫺-contaminated water. Indian mustard and muskgrass were efficient in degrading SeCN⫺ because XAS revealed that none of
the Se accumulated in the tissues of plants treated
with SeCN⫺ remained in the form of SeCN⫺. Thus,
plants have good potential for the detoxification of
SeCN⫺, regardless of whether they are an upland or
wetland species.
A Possible Mechanism for the Assimilation and
Phytodegradation of SeCN
Indian mustard and muskgrass were able to remove the cyanide moiety of SeCN⫺ and convert the
Se to organic Se and less toxic DMSe. The biochemical processes that are responsible for the accumulation and assimilation of SeCN⫺ to volatile Se have
not yet been elucidated.
Although cyanogenesis is well studied because of
the toxicological role of hydrogen cyanide production from cyanogenic glycosides in some food plants
(Jones, 1998; Vetter, 2000), OCN⫺ degradation has
received little attention. The Brassicaceae are not cyanogenic, but they produce thiocyanate, the chemical
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
629
de Souza et al.
analog of SeCN⫺, as a toxic byproduct of glucosinolate hydrolysis (Fenwick et al., 1983; Angus et al.,
1994; Brown and Morra, 1996). The thiocyanate is
believed to act as a plant defense mechanism during
attack by fungal pathogens or insect pests (Fenwick
et al., 1983; Angus et al., 1994). Sulfur from thiocyanate may enter the sulfur assimilation pathway to
produce other volatile sulfur gases, e.g. dimethylsulfide (Forney and Jordan, 1998). Thus, it is possible
that plants use the biochemical pathways for the
conversion of thiocyanate to dimethylsulfide for the
assimilation of SeCN⫺ to DMSe. Indian mustard has
been shown to assimilate selenate via the sulfate
assimilation pathway (Pilon-Smits et al., 1999; Terry
et al., 2000). Therefore, a pathway of SeCN⫺ metabolism was proposed in analogy to thiocyanate metabolism in plants (Fig. 5).
The first step in the proposed SeCN⫺ assimilation
pathway is the uptake of SeCN⫺. Given the rapid
accumulation of Se and cyanide into Indian mustard
seedlings and the high Se concentrations accumulated in tissue of mature plants after 5 d of treatment,
it is possible the SeCN⫺ is accumulated actively in a
manner similar to selenate (Leggett and Epstein,
1956) and Se-Met (Abrams et al., 1990).
In the second step, SeCN⫺ may be degraded to
Se2⫺ and OCN⫺ by an unknown enzyme, as suggested for bacteria (Youatt, 1954; Happold et al.,
1958). OCN⫺ is then degraded to ammonia and CO2
Figure 5. Proposed pathway of SeCN⫺ assimilation by Indian mustard to the volatile Se forms DMSe and H2Se. Also shown is a
simplified version of the selenate assimilation pathway for Indian
mustard (Terry et al., 2000). The numbers by the arrows represent the
enzymes involved. The two major rate-limiting enzymes for selenate
assimilation to DMSe are ATP sulfurylase (1) and Met methyltransferase (MMT, 2). Enzyme 3 is a novel thiol methyltransferase (Attieh
et al., 2000) that methylates SeCN⫺ to CH3SeCN. Enzyme 4 is
unknown or a plant homolog for the bacterial thiocyanate hydrolase.
This enzyme degrades SeCN⫺ to volatile H2Se or the Se2⫺ that enters
the Se assimilation pathway for DMSe production. OCN⫺ is detoxified by the enzyme cyanase (5), which has been cloned from Arabidopsis (Aichi et al., 1998).
630
by the enzyme cyanase. The gene encoding cyanase
was recently cloned in Arabidopsis (Aichi et al.,
1998), which is a member of the Brassicaceae. In an
alternate manner, plants could produce Se2⫺ via an
alternative pathway of thiocyanate degradation,
where the enzyme thiocyanate hydrolase mediates
the degradation of thiocyanate to ammonia and carbonylsulfide (Katayama et al., 1992, 1993, 1998). The
carbonylsulfide is then degraded to S2⫺ and CO2 by
an unknown enzyme.
In the third step, Se2⫺ enters the pathway described
for the assimilation of inorganic Se to DMSe (Terry et
al., 2000). The incorporation of Se2⫺ into an amino
acid backbone provided by O-acetyl-Ser results in the
production of seleno-Cys (Ng and Anderson, 1978),
which is likely to serve as a precursor of Se-Met
(Terry et al., 2000). The XAS data is consistent with
this proposed pathway because the Se accumulated
by SeCN⫺-supplied Indian mustard plants was
mainly in the form of organic Se forms with no Se
remaining in the form of SeCN⫺ (Fig. 4). Se-Met is
thought to be methylated by MMT to methylSe-Met,
which is cleaved to form DMSe. It was recently
shown that MMT is a key enzyme for DMSe production by Arabidopsis plants treated with different
forms of Se (A. Tagmount, A. Berken, and N. Terry,
unpublished data).
The GC/MS analysis identified DMSe as the major
volatile Se form produced by SeCN⫺-treated Indian
mustard. However, the production of volatile
CH3SeCN could not be ruled out. Therefore, the
pathway of SeCN⫺ metabolism in Figure 5 includes
its methylation to CH3SeCN in a manner similar to
the detoxification of thiocyanate by a novel thiol
methyltransferase produced by cabbage (Brassica oleracea capitata L.; Attieh et al., 2000).
Indian mustard plants accumulated large amounts
of organic Se in their tissues when supplied with
SeCN⫺ (29% [w/v] of the Se supplied), but they did
not produce large amounts of volatile Se (0.7% [w/v]
of the SeCN⫺ removed was in the form of volatile Se).
It is very likely that one or more biochemical steps in
the conversion of organic Se to DMSe is rate limiting,
e.g. the methylation of Se-Met by MMT. Transgenic
Indian mustard plants that overexpress MMT may
overcome the rate limitation and produce more
DMSe when treated with SeCN⫺.
In conclusion, plants such as Indian mustard are
promising for the phytoremediation of SeCN⫺contaminated soil and water because of their remarkable abilities to phytoextract SeCN⫺ and degrade all
the accumulated SeCN⫺ to other Se forms. Furthermore, the tolerance of plants to toxic levels of SeCN⫺
was similar to or higher than other forms of Se and
they were able to convert some of the SeCN⫺ to less
toxic DMSe, which may be removed from the contaminated site into the atmosphere.
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 128, 2002
Se Assimilation and Volatilization from Selenocyanate
MATERIALS AND METHODS
Seeds of Indian mustard (Brassica juncea; accession no.
173874) were obtained from the North Central Regional
Plant Introduction Station (Ames, IA). The ability of Indian
mustard to take up SeCN⫺ was determined. Indian mustard seeds were surface sterilized by treatment with 70%
(w/v) ethanol for 30 s and 20% (w/v) bleach for 30 min,
followed by five washes with sterile water. During these
treatments, the seeds were kept in sterile plastic tubes that
were closed tightly and kept on a rocking platform to
ensure equal access of the bleach or alcohol to all the seeds.
After blotting the seeds on sterile filter paper in a laminar
flow hood, they were transferred into sterile Magenta
boxes containing 50 mL of sterile one-half-strength Hoagland solution (Hoagland and Arnon, 1938) solidified with
0.4% (w/v) Phytagar (Invitrogen, Carlsbad, CA). The boxes
were placed under constant light in a controlled environment room that was maintained at 25°C and 40% humidity.
After 1 week of growth, a solution of 100 ␮m SeCN⫺ was
added to the boxes, overlaying the agar. After 30 min, the
seedlings were gently removed with forceps, washed in
running deionized water, and dried at 55°C overnight.
One-half of the samples were ground, weighed, and digested for Se analysis as described below. The rest of the
samples were analyzed for their total cyanide content by
Ana-Lab (Kilgore, TX) using EPA method 9012.
In another experiment, the role of bacteria in SeCN⫺
accumulation by Indian mustard was determined. Seeds
were surface sterilized as described above. One batch of
axenic seeds was inoculated with three randomly selected
strains of bacteria that we had isolated previously from the
rhizosphere of Indian mustard (de Souza et al., 1999). The
three strains (BJ1, BJ5, and BJ10) were identified as close
relatives of Microbacterium saperdae, Pseudomonas monteilii,
and Enterobacter cancerogenes, based on ⬎99% similarity in
their 16S rDNA sequences (M.P. de Souza and N. Terry,
unpublished data). A mixed inoculum of these three strains
was prepared to add to the seeds: one loopful of each
bacterial strain was added to 5 mL of sterile 0.85% (w/v)
saline (NaCl) solution in a sterile plastic tube and vortexed
to suspend the bacteria. One milliliter of this suspension
was added to 4 mL of sterile 0.5% (w/v) methylcellulose
solution (Sigma, St. Louis) prepared with 0.85% (w/v)
sterile saline solution, and one batch of axenic seeds were
soaked in this methylcellulose solution containing bacteria.
An additional tube containing a second batch of seeds and
2.5 mL of sterile methylcellulose provided an axenic control. After 20 min of soaking time, all seeds were removed
from the methylcellulose solutions and dried on sterile
filter papers in a laminar flow cabinet. The seeds were
sown in Magenta boxes containing 50 mL of one-halfstrength Hoagland solution containing 0.4% (w/v) Phytagar (Invitrogen) and 0.22-␮m filter-sterilized KSeCN (20
␮m), which was added just before the media solidified. The
boxes were placed under constant light in a controlled
environment growth room, and were harvested after 10 d.
At harvest, the seedlings were removed from the boxes,
rinsed thoroughly in deionized water to remove adhering
agar, dried at 80°C, and weighed. The concentrations of Se
Plant Physiol. Vol. 128, 2002
in the seedlings were determined after acid digestion and
analysis by hydride-generation atomic absorption spectroscopy, as described below. To determine the role of microbes in SeCN⫺ assimilation, a separate batch of Indian
mustard seedlings was grown similarly, and after harvest,
the fresh tissues from axenic and bacteria-treated seedlings
were ground in liquid nitrogen. These tissues were then
frozen at ⫺80°C for XAS analysis as described below.
To determine the tolerance of Indian mustard seedlings
to SeCN⫺, seeds were surface sterilized as described above
and sowed in Magenta boxes containing 50 mL of one-halfstrength Murashige and Skoog agar medium (Sigma), 1%
(w/v) Suc, and 0.4% (w/v) Phytagar (pH 5.8). After the
media had been autoclaved and cooled, sodium selenate,
sodium selenite, or KSeCN were added from 0.22-␮m
filter-sterilized stock solutions to make triplicate Magenta
boxes containing 20 and 200 ␮m Se for each Se species.
Eight seeds were sowed in each Magenta box. The boxes
were incubated in a controlled environment growth room
under constant light. After 7 d, the seedlings were gently
removed from the agar, washed to remove any agar sticking to the roots, and the root lengths and fresh weights of
individual seedlings were measured.
For the mature plant experiments, Indian mustard seeds
were germinated on moistened filter paper and transferred
after 2 d into 3.5-inch pots containing coarse sand. The pots
were placed in flats that were filled halfway to the top with
one-half-strength Hoagland solution and they were maintained in a greenhouse with controlled temperature (25°C–
30°C) and a long-day (16-h) photoperiod. The plants were
watered twice a day, once with tap water and once with
one-half-strength Hoagland solution. After 2 weeks, the
plants were carefully removed from the sand to avoid
damage to the roots, washed in deionized water, and
placed in containers containing 18 L of one-half-strength
Hoagland solution for 10 d. The hydroponic solutions were
aerated.
Muskgrass (Chara canescens) was collected from the Allegheny Power Services constructed wetland (Springdale,
PA; Ye et al., 2001), washed with deionized water, and
cultured with aerated one-half-strength Hoagland solution
in the greenhouse for 1 week. After 1 week in hydroponic
solution, five replicates each of Indian mustard and
muskgrass were transferred into one-half-strength Hoagland solutions containing 20 ␮m Se supplied as sodium
selenate, sodium selenite, or KSeCN (Sigma). Conditions of
1 week pretreatment with 20 ␮m Se were chosen because
the kinetics of Se accumulation and Se volatilization by
Indian mustard was linear at concentrations between 0.2
and 200 ␮m Se and up to 14 d when treated with 20 ␮m Se
(de Souza et al., 1998).
After 1 week on Se, one replicate plant from each treatment was washed thoroughly in running deionized water.
The muskgrass tissue and the roots and shoots of Indian
mustard were ground separately in liquid nitrogen. These
tissues were then frozen at ⫺80°C for XAS analysis as
described below.
Se volatilization was measured from the other four replicates of each treatment by placing the Indian mustard
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
631
de Souza et al.
plants or the muskgrass in Magenta boxes (Sigma) containing 200 mL of one-half-strength Hoagland solution and 20
␮m selenate, selenite, or SeCN⫺. The Magenta boxes were
placed in gastight acrylic volatilization chambers (3-L volume) through which a continuous air flow (1.5 L min⫺1)
was passed by applying suction at the outlet and by bubbling incoming air into the hydroponic solution. Volatile Se
was quantitatively trapped in alkaline peroxide liquid
traps as described previously (Zayed and Terry, 1992). The
Se volatilization chambers were placed in the greenhouse.
Aliquots (10 mL) of trap solution were collected every 24 h,
after which the solutions were replaced. The trap solution
samples were heated at 95°C to remove the peroxide. The
selenate-Se was reduced to selenite by adding an equal
volume of concentrated HCl and heating at 95°C for 30
min. The Se concentration was measured by vaporgeneration atomic absorption spectroscopy as described
below.
In a separate experiment, GC/MS was used to determine the form of volatile Se given off by Indian mustard.
Briefly, axenic seedlings of Indian mustard were grown in
15-mL glass serum vials (Fisher Scientific, Pittsburgh)
containing 20 ␮m SeCN⫺, selenate, or selenite. The vials
were closed with Teflon-faced butyl rubber stoppers
(Wheaton, Millville, NJ), crimp sealed, incubated in a
growth chamber for 7 d, and frozen at ⫺20°C. After
thawing, the gas phase was sampled (100 ␮L) with a
1710SL gas tight syringe (Hamilton, Reno, NV) and injected into the GC with the injection port split flow off for
10 s then on at 20 mL min⫺1 thereafter. GC/MS analyses
were run on a 4500 mass spectrometer (Thermo Finnigan,
San Jose, CA). A 30-m Rtx-5 column (Restek, Bellefonte,
PA) was used to separate volatile compounds in the headspace. The column oven was maintained at room temperature for 3 min and was then ramped at 10°C min⫺1 to
125°C. The mass spectrometer was scanned in selected ion
mode looking at two ions for each analyte, e.g. 80 and 82
for H2Se, 94 and 96 for methane selenol (CH3SeH), 107
and 109 for methylselenocyanate (CH3SeCN), 95 and 110
for DMSe, and 188 and 190 for DMDSe. Known standards
of DMDSe (Aldrich Chemical, Milwaukee, WI) and DMSe
(Alfar Aesar, Ward Hill, MA) were also run along with the
samples. There are no commercially available standards
for H2Se, CH3SeH, and CH3SeCN, which are three other
potential volatile Se forms that may be produced from
SeCN⫺.
After measuring Se volatilization for 5 d, the Indian
mustard or muskgrass was washed thoroughly in running
water to remove any Se adhering to the tissue, and was
dried at 55°C for 3 d. Roots and shoots of Indian mustard
and whole tissue of muskgrass were weighed and ground
separately using a Wiley mill. The tissues were digested by
stepwise additions of 70% (w/v) nitric acid, 30% (w/v)
hydrogen peroxide, and concentrated HCl at 95°C in a
modification of EPA protocols 3052 B and 7742 (Bañuelos
and Pflaum 1990). A wheat flour standard (National Institute of Science and Technology, 1.1 mg of Se kg⫺1) and a
blank were used with all digestions. The Se content in the
acid digests of plant tissues or in the acid-treated trap
632
solution was measured by hydride-generation atomic absorption spectroscopy (Martin, 1975). The background Se
concentrations were measured in untreated plants and
were subtracted from the values obtained for plants treated
with Se. The detection limit of this analytical method was 1
␮g of Se L⫺1. Selenium dioxide reference solution (Fisher
Scientific) was diluted in 6 m HCL and used as a standard.
All samples were diluted in 6 m HCl to give absorbances in
the linear portion of the standard curve.
The forms of Se accumulated in the frozen samples of
seedlings and mature plants were determined by XAS at
the Stanford Synchrotron Radiation Laboratory on Beam
Line 4–3. Frozen samples were positioned in a liquid He
cryostat at a 45° angle to the x-ray beam. A Si(220) doublecrystal monochromator was used with an upstream vertical aperture of 1 mm, and harmonic rejection was
achieved by detuning one crystal by 50%. The electron
energy was 3.0 GeV with a current of ⬇50 to 100 mA.
Selenium K-edge x-ray absorption spectra were collected
by monitoring the Se K␣ fluorescence using a Canberra
13-element Ge detector in a series of replicate scans dependent on trace element concentration. Spectra were also
collected for reference solutions, i.e. 10 mm solutions of
sodium selenate, sodium selenite, potassium SeCN⫺, SeMet, and selenocystine (Sigma). All samples were calibrated against a reference of hexagonal Se(0) collected
simultaneously with the data in transmission; the first
energy-inflection of the reference was assumed to be
12658.0 eV. Data were collected using the program XASCollect (George, 2000) and were analyzed using the
EXAFSPAK suite of programs (http://ssrl.slac.
stanford.edu/exafspak.html). Quantitative analysis using
an edge-fitting method was carried out according to the
method of Pickering et al. (1995).
Statistical analyses were performed using the JMP IN
statistical package (SAS Institute, Cary, NC) using analysis
of variance procedures.
ACKNOWLEDGMENTS
We thank Danika LeDuc for helpful comments on the
manuscript, Patricia Fox, Marina Ma, May Zhao, and
Morgan Bauer for technical assistance, Zhiqing Lin for
collecting the Chara, and the staff of the University of
California (Berkeley) herbarium for help with algal
identification.
Received August 2, 2001; returned for revision October 3,
2001; accepted November 7, 2001.
LITERATURE CITED
Abrams MM, Shennan C, Zasoski RJ, Burau RG (1990)
Selenomethionine uptake by wheat seedlings. Agron J
82: 1127–1130
Aichi M, Nishida I, Omata T (1998) Molecular cloning and
characterization of a cDNA encoding cyanase from Arabidopsis thaliana. Plant Cell Physiol Suppl 39: S135
Angus JF, Gardner PA, Kirkegaard JA, Desmarchelier JM
(1994) Biofumigation: isothiocyanates released from
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 128, 2002
Se Assimilation and Volatilization from Selenocyanate
Brassica roots inhibit growth of the take-all fungus. Plant
Soil 162: 107–112
Attieh J, Kleppinger-Sparace KF, Nunes C, Sparace SA,
Saini HS (2000) Evidence implicating a novel thiol methyltransferase in the detoxification of glucosinolate hydrolysis products in Brassica oleracea L. Plant Cell Environ 23: 165–174
Bañuelos G, Pflaum T (1990) Determination of selenium in
plant tissue with optimal digestion conditions. Commun
Soil Sci Plant Anal 21: 1717–1726
Bañuelos GS, Meek DW (1990) Accumulation of selenium
in plants grown on selenium-treated soil. J Environ Qual
19: 772–777
Bañuelos GS, Terry N, Zayed A, Wu L (1995) Managing
high soil Se with phytoremediation. In GE Schuman, GF
Vance, eds, Selenium, Mining, Reclamation, and Environmental Impact. American Society of Surface Mining
and Reclamation, Gillete, WY, pp 394–405
Brown PD, Morra MJ (1996) Hydrolysis products of glucosinolates in Brassica napus tissues as inhibitors of seed
germination. Plant Soil 181: 307–316
Carneiro MER, Azevedo C, Ramalho NM, Knoppers B
(1994) The biomass of Chara hornemannii in relation to the
physical and chemical conditions of the Piratininga lagoon. An Acad Bras Cienc 66: 213–222
de Souza MP, Chu D, Zhao M, Zayed A, Ruzin SE,
Schichnes D, Terry N (1999) Rhizosphere bacteria enhance Se accumulation and volatilization by Indian mustard (Brassica juncea L.). Plant Physiol 119: 565–573
de Souza MP, Pilon-Smits EAH, Lytle CM, Hwang S, Tai
J, Honma TSU, Yeh L, Terry N (1998) Rate limiting steps
in Se assimilation and volatilization by Brassica juncea.
Plant Physiol 117: 1487–1494
Dushkenov V, Kumar PBAN, Motto H, Raskin I (1995)
Rhizofiltration: the use of plants to remove heavy metals
from aqueous streams. Environ Sci Technol 29: 1239–1245
Fenwick GR, Heaney RK, Mullin WT (1983) Glucosinolates and their breakdown products in food and food
plants. CRC Crit Rev Food Sci Nutr 18: 123–201
Forney CF, Jordan MA (1998) Induction of volatile compounds in broccoli by postharvest hot-water dips. J Agric
Food Chem 46: 5295–5301
George MJ (2000) XAS-Collect: a computer program for
X-ray absorption spectroscopic data acquisition. J Synchrotron Rad 7: 283–286
Hansen D, Duda P, Zayed AM, Terry N (1998) Selenium
removal by constructed wetlands: role of biological volatilization. Environ Sci Technol 32: 591–597
Happold FC, Jones GL, Pratt DB (1958) Utilization of
thiocyanate by Thiobacillus thioparus and T. thiooxidans.
Nature 182: 266–267
Herrera-Silveira JA (1994) Phytoplankton productivity
and submerged macrophyte biomass variation in a tropical coastal lagoon with ground water discharge. Vie et
Mileu 44: 257–266
Hoagland D, Arnon DI (1938) The water culture method
for growing plants without soil. Bull Calif Agric Stat 346
Horne AJ (2000) Phytoremediation by constructed wetlands. In N Terry, G Bañuelos, eds, Phytoremediation of
Plant Physiol. Vol. 128, 2002
Contaminated Soil and Water. Lewis Publishers, New
York, pp 13–39
Jones DA (1998) Why are so many food plants cyanogenic?
Phytochemistry 47: 155–162
Katayama Y, Kanagawa T, Kuraishi H (1993) Emission of
carbonyl sulfide by Thiobacillus thioparus grown with
thiocyanate in pure and mixed cultures. FEMS Microbiol
Lett 114: 223–228
Katayama Y, Narahara Y, Inoue Y, Amano F, Kanagawa T,
Kuraishi H (1992) A thiocyanate hydrolase of Thiobacillus
thioparus: a novel enzyme catalyzing the formation of carbonyl sulfide from thiocyanate. J Biol Chem 267: 9170–9175
Katayama Y, Narahara Y, Inoue Y, Amano F, Kanagawa T,
Kuraishi H (1998) Cloning of genes coding for the subunits of thiocyanate hydrolase of Thiobacillus thioparus
THI 115 and their evolutionary relationships to nitrile
hydratase. J Bacteriol 180: 2583–2589
Kumar PBAN, Dushkenov V, Motto H, Raskin I (1995)
Phytoextraction: the use of plants to remove heavy metals from soils. Environ Sci Technol 29: 1232–1238
Leggett JE, Epstein E (1956) Kinetics of sulfate adsorption
by barley roots. Plant Physiol 31: 222–226
Lin ZQ, Schemenauer RS, Cervinka V, Zayed A, Lee A,
Terry N (2000) Selenium volatilization from a soil-plant
system for the remediation of contaminated water and soil
in the San Joaquin Valley. J Environ Qual 29: 1048–1056
Manceau A, Gallup DL (1997) Removal of selenocyanate in
water by precipitation: characterization of copper-selenium
precipitate by x-ray diffraction, infrared, and x-ray absorption spectroscopy. Environ Sci Technol 31: 968–976
Martin TD (1975) Determining selenium in wastewater
sediment and sludge by flameless atomic absorption.
Atomic Abs Newslett 14: 109–116
Ng BH, Anderson JW (1978) Synthesis of selenocysteine by
cysteine synthases from selenium accumulator and nonaccumulator plants. Phytochemistry 18: 573–580
Pickering IJ, Brown GE Jr, Tokunaga TK (1995) Quantitative speciation of selenium in soils using X-ray absorption spectroscopy. Environ Sci Technol 29: 2456–2459
Pilon-Smits EAH, Hwang S, Lytle CM, Zhu Y, Tai JC,
Bravo RC, Chen Y, Leustek T, Terry N (1999) Overexpression of ATP sulfurylase in Indian mustard leads to
increased selenate uptake, reduction, and tolerance.
Plant Physiol 119: 123–132
Terry N, Zayed AM, de Souza MP, Tarun AS (2000) Selenium in higher plants. Annu Rev Plant Physiol Plant Mol
Biol 51: 401–432
Vetter J (2000) Plant cyanogenic glycosides. Toxicon 38:
11–36
Wilber CG (1980) Toxicology of selenium: a review. Clin
Toxicol 17: 171–230
Ye ZH, Whiting SN, Lin ZQ, Lytle CM, Qian JH, Terry N
(2001) Removal and distribution of Fe, Mn, Co, and Ni
within a constructed wetland treating coal combustion
by-product leachate. J Environ Qual 30: 1464–1473
Youatt JB (1954) Studies on the metabolism of Thiobacillus
thiocyanooxidans. J Gen Microbiol 11: 139–149
Zayed A, Terry N (1992) Selenium volatilization in broccoli
as influenced by sulfate supply. J Plant Physiol 140:
646–652
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
633