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Overexpression of ATP Sulfurylase in Indian Mustard: Effects on Tolerance and

Accumulation of Twelve Metals

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DOI: 10.2134/jeq2004.5400 · Source: PubMed

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 Overexpression of ATP Sulfurylase in Indian Mustard: Effects on Tolerance
and Accumulation of Twelve Metals
Ami L. Wangeline, Jason L. Burkhead, Kerry L. Hale, Stormy D. Lindblom, Norman Terry,
Marinus Pilon, and Elizabeth A. H. Pilon-Smits*

ABSTRACT zation by plant roots (Berti and Cunningham, 2000). In

Indian mustard [Brassica juncea (L.) Czern.] transgenics overex-
pressing ATP sulfurylase (APS plants) were shown previously to have
higher levels of total thiols, S, and Se. The present study explores the
effect of ATP sulfurylase overexpression on tolerance and accumula-
tion of other metals, both oxyanions and cations, reasoning that some
anions may react directly with ATP sulfurylase, while other ions
may be bound by its thiol end products. The APS transgenics were
compared with wild-type plants with respect to tolerance and accumu-
lation of As, Cd, Cr, Cu, Hg, Mn, Mo, Ni, Pb, V, W, and Zn, supplied
individually in agar medium (seedlings) or in hydroponics (mature
plants). At the seedling stage, APS transgenics were more tolerant
than wild type to As(III), As(V), Cd, Cu, Hg, and Zn, but less tolerant
to Mo and V. The APS seedlings had up to 2.5-fold higher shoot
the technology called rhizofiltration, accumulation of
metals by roots in a hydroponic setup is followed by
harvesting of the plant biomass (Dushenkov and Kapul-
nik, 2000). Phytoextraction, another promising technol-
ogy in metal phytoremediation, involves the accumula-
tion of metals in shoot tissue followed by harvesting of
the shoot biomass (Blaylock and Huang, 2000). The
metal-laden plant material may be used for nonfood
purposes or ashed, followed by recycling of the metals
or disposal in a landfill (Chaney et al., 2000). These
metal phytoremediation technologies are already being
used effectively (Salt et al., 1998; Blaylock, 2000), and

f
concentrations of As(III), As(V), Hg, Mo, Pb, and V, and somewhat are gaining acceptance, since phytoremediation is rela-

lower Cr levels. Mature APS plants contained up to 2.5-fold higher
shoot concentrations of Cd, Cr, Cu, Mo, V, and W than wild type. They
also contained 1.5- to 2-fold higher levels of the essential elements Fe,
Mo, and S in most of the treatments. Mature APS plants showed no
oo
tively cost-effective and aesthetically pleasing, and may
be used in conjunction with more conventional remedia-
tion methods.
To further increase the effectiveness of metal phytor-

Pr
differences in metal tolerance compared with the wild type. Overex-
pression of ATP sulfurylase may be a promising approach to create
emediation, several approaches may be employed, in-
plants with enhanced phytoextraction capacity for mixtures of metals.
cluding identification of new suitable plant species via
screening studies, optimization of agronomic practices
for maximal element uptake, and improvement of se-
ed
lected plant species by classical breeding or genetic engi-
T oxic metals and metalloids are increasingly re-
leased into the environment by human activities
such as industry, mining operations, use of ammunition,
neering.
Genetic engineering is starting to emerge as a rela-
ct

tively rapid and effective way to improve the capacity

traffic, and agriculture, resulting in contamination that
of plants to tolerate and accumulate metals. Transgenic
threatens natural ecosystems and human well-being
plants with enhanced metal tolerance and accumulation
re

(Lantzy and Mackenzie, 1979; Nriagu, 1979; Ross, 1994).

have been created through several approaches, includ-
Currently, the USA is spending around $3 billion a year
ing overexpression of metal transporter proteins (Sam-
on remediation of toxic inorganic elements, comprising
or

uelsen et al., 1998; Arazi et al., 1999; Van der Zaal et

35% of the total U.S. funds spent for environmental
al., 1999; Curie et al., 2000; Hirschi et al., 2000), overpro-
cleanup (Glass, 1999, 2000). Some examples of conven-
duction of metal-chelating molecules (Evans et al., 1992;
tional remediation methods for metals include soil wash-
nc

de la Fuente et al., 1997; Hasegawa et al., 1997; Goto

ing, excavation and reburial of the soil, and soil stabiliza-
et al., 1999; Zhu et al., 1999a, 1999b), or even introduc-
tion by, for example, concrete capping.
tion of a bacterial pathway (Rugh et al., 1996; Bizily et
Alternative phytoremediation methods for metals
U

al., 1999, 2000). For a review of the development of

and metalloids make use of the natural ability of plants
to acquire minerals from their environment and to stabi-
lize soil and create an upward water flow in the process.
Phytostabilization may involve the prevention of leach-
ing through an upward water flow resulting from plant
transpiration, reduced runoff owing to aboveground
vegetation, and reduced soil erosion through soil stabili-
A.L. Wangeline, J.L. Burkhead, K.L. Hale, S.D. Lindblom, M. Pilon,
and E.A.H. Pilon-Smits, Biology Department, Colorado State Univer-
sity, A/Z Building, Fort Collins, CO 80523. N. Terry, Department
of Plant and Microbial Biology, 111 Koshland Hall, University of
California, Berkeley, CA 94270. Received 14 Oct. 2002. *Correspond-
ing author (epsmits@lamar.colostate.edu).
Published in J. Environ. Qual. 33:䊏–䊏 (2004).
 ASA, CSSA, SSSA
677 S. Segoe Rd., Madison, WI 53711 USA
transgenics for metal phytoremediation, see Krämer and
Chardonnens (2001) and Pilon-Smits and Pilon (2002).
Phytochelatins are cysteine-rich metal-chelating pep-
tides involved in heavy metal tolerance and sequestra-
tion (Steffens, 1990). The general structure of phyto-
chelatins is (␥-Glu-Cys)n–Gly, where n ⫽ 2 to 11
(Rauser, 1995). The cysteine needed for the biosynthesis
of phytochelatins is produced by the sulfate assimilation
pathway, which is located primarily in the chloroplast
in plants (Schwenn, 1994; Leustek, 1996; Leustek and
Saito, 1999). After uptake by sulfate permease, sulfate
is activated by ATP sulfurylase to form adenosine phos-
phosulfate, which is subsequently reduced to free sulfite
Abbreviations: APS, plants overexpressing ATP sulfurylase; WT,
wild type.

1
2 J. ENVIRON. QUAL., VOL. 33, JANUARY–FEBRUARY 2004

by APS reductase (Setya et al., 1996). Sulfate permease Table 1. Elements and concentrations used for treatments of
and ATP sulfurylase are induced by sulfur starvation seedlings and mature plants.
and repressed by feeding sulfate or reduced forms of S Concentration Concentration
Chemical supplied supplied to
(Chen and Leustek, 1995; Logan et al., 1996). Element form supplied to seedlings mature plants
Overexpression of ATP sulfurylase in Indian mustard
mg L⫺1
APS transgenics resulted in twofold higher levels of the
As(III) NaAsO2 4 3
organic sulfur compounds glutathione and total nonpro- As(V) Na2HAsO4 25 –
tein thiols, as well as higher total sulfur levels (Pilon- Cd CdSO4 20 7.5
Smits et al., 1999). The transgenic APS plants also Cr K2CrO4 10 4.5
Cu CuSO4 5 2
showed enhanced tolerance to selenium (threefold), in- Hg HgCl2 8 2
creased reduction of selenate to organic forms of sele- Mn MnCl2 75 50
Mo (NH4)6Mo7O24 85 40
nium, and higher selenium accumulation (Pilon-Smits Ni NiSO4 10 7.5
et al., 1999). These results, obtained using hydroponics, Pb Pb(NO3)2 75 25
show that ATP sulfurylase is involved in selenate as V Na2VO4 10 7
W Na2WO4 100 50
well as sulfate assimilation, and is probably present in Zn ZnSO4 40 10
wild-type Indian mustard in amounts that are rate-lim-
iting for the uptake and reduction of sulfur and sele-
nium. The observed enhanced selenium tolerance and (Ames, IA). Transgenic APS seeds were obtained from plants
accumulation in the APS transgenics suggest they may of this same accession number, genetically engineered to over-
have enhanced selenium phytoremediation capacity. In- express ATP sulfurylase (APS). The transgenic APS plants

f
overexpress the Arabidopsis thaliana APS1 cDNA including
deed, the APS plants showed threefold higher shoot

selenium levels compared with wild type when grown
on soil naturally rich in selenium (unpublished results).
This suggests that hydroponic systems are a useful
oo
its own chloroplast transit sequence, under the control of the
CaMV 35S promoter (Pilon-Smits et al., 1999). The transgenic
APS line used for this study was APS8, bred to homozygosity.
The chemical forms and concentrations of the metals and

means to explore plants’ phytoremediation capacity and
that overexpression of ATP sulfurylase is a promising
approach to create transgenic plants with enhanced sele-
nium phytoremediation capacity.
ed
The objective of the present study was to conduct a
more comprehensive analysis of metal tolerance and
accumulation of the APS transgenics relative to wild-
ct
Pr
metalloids used are shown in Table 1. These chemical forms
were chosen to have high solubility in water and to have
minimal effect on the nutrient solution composition. No pre-
cipitation was observed when these metal salts were added to
agar medium or nutrient solution. The concentrations used
were aimed to give approximately 50% reduction in plant
growth (on a fresh weight basis) compared with untreated
controls, a degree of toxicity expected to optimally reveal any

type Indian mustard, with the combined goal to obtain

differences in tolerance between the APS transgenics and the
more insight into the involvement of this enzyme in wild type (WT).
plant metal tolerance and accumulation, and to further
re

explore the phytoremediation potential of the APS

transgenics. For instance, it is feasible that the APS Metal Tolerance and Accumulation Experiments
transgenics will show higher accumulation of other oxy-
or

Seedlings
anions that are similar to sulfate and selenate, such as
molybdate, tungstate, chromate, vanadate, and arse- To compare the metal tolerance of APS and WT Indian
nc

nate, if the ATP sulfurylase enzyme can use these ions
as a substrate. Also, the higher thiol levels in the APS
transgenics may confer enhanced tolerance and accumu-
lation of thiol-bound metals such as cadmium, as was
U
mustard plants at the seedling level, WT and APS seedlings
were grown axenically from seed on agar medium containing
one of the selected metals. For each experiment, a treatment
without added metal was run in parallel as a control. The

seeds were surface-sterilized as described by Pilon-Smits et

found for other thiol-overproducing transgenics (Zhu
et al., 1999a, 1999b). Promising in this respect was the
observation that APS transgenics removed more Cd,
Cr, Cu, Mn, Pb, and Zn from polluted soil in comparison
with wild-type Indian mustard (Bennett et al., 2002).
To explore the role of ATP sulfurylase in plant tolerance
and accumulation of various metals and metalloids, and
the potential of APS plants for phytoremediation of
these elements, controlled experiments were done to
compare APS and wild-type plants with respect to their
accumulation and tolerance of the 12 toxic elements As,
Cd, Cr, Cu, Hg, Mn, Mo, Ni, Pb, V, W, and Zn.
MATERIALS AND METHODS
Materials
Indian mustard wild-type seeds were from Accession no.
173874, North Central Regional Plant Introduction Station
al. (1999), and 36 sterilized seeds were sown in a grid pattern
on agar medium in Magenta tissue culture boxes (Sigma, St.
Louis, MO). The agar medium contained half-strength Muras-
hige and Skoog (MS) salts and vitamins (Murashige and
Skoog, 1962), 10 g L⫺1 sucrose, and 4 g L⫺1 agargel (Sigma);
individual metals were added to the final concentrations
shown in Table 1. These concentrations were aimed to give
an approximately 50% reduction in wild-type seedling fresh
weight, corresponding with an approximately 75% reduction
in seedling root length compared with untreated controls. The
magenta boxes with the different treatments were randomly
arranged and the seedlings were allowed to grow for 7 d in a
growth cabinet at 25⬚C and a 16 h light–8 h dark photoperiod.
Individual seedlings were then harvested and washed, and
root length was measured as a parameter for metal tolerance
(Murphy and Taiz, 1995). To correct for any differences be-
tween experiments, metal tolerance was expressed as relative
root length, calculated as root length in the presence of a
metal divided by root length on control medium.
 WANGELINE ET AL.: OVEREXPRESSION OF ATP SULFURYLASE IN INDIAN MUSTARD
Mature Plants
To compare metal tolerance at the mature plant level, seeds
of WT and APS transgenics were surface-sterilized, sown on
agar medium as described above, and grown for 4 d. The
seedlings were then transferred to sand, watered with half-
strength Hoagland’s nutrient solution daily (Hoagland and
Arnon, 1938), and grown in a greenhouse at 25⬚C, 16 h light–8 h
dark photoperiod until they were 35 d old. At this point, the
plants were transferred to a greenhouse nutrient film tech-
nique setup as described by Zhu et al. (1999a). After 7 d of
adjustment to the new medium, the fresh weights of the plants
were measured and the individual metal treatments were
started. One metal was given at a time at the concentrations
shown in Table 1; for each experiment a control treatment
without metal was run in parallel. These metal concentrations
were expected to give an approximately 50% reduction in
fresh weight in mature plants compared with untreated con-
trols. Ten replicate plants were used per plant genotype (WT
or APS) per treatment. The nutrient solutions were replaced
every 3 d for a total of 14 d, after which the plants were
harvested, washed, weighed, and dried for elemental analysis.
Elemental Analysis
Seedlings
Seedling shoot tissue was collected and dried for 48 h at
80⬚C for elemental analysis. Per treatment, five dried shoot
samples of six or seven seedlings each were weighed and acid-
digested for 6 h at 130⬚C in concentrated nitric acid according
to the method of Zarcinas et al. (1987). After dilution of the
acid digests with distilled water the total elemental concentra-
tions in the digests were measured using inductively coupled
ct
plasma–atomic emission spectrometry (ICP–AES; Thermo
Elemental, Franklin Lakes, NJ) according to the method of
Fassel (1978), using appropriate standards and quality controls.
re
Mature Plants
or
After harvesting, the plants were dried for 48 h at 80⬚C.
Each individual shoot was ground in a Wiley mill and homoge-
nized, and a sample was taken for acid digestion and analysis
nc
by ICP–AES as described above.
Statistical Analyses
U
Statistical analyses for tolerance and accumulation were
performed using the software program JMP-IN (SAS Institute,
1999). Statistically significant differences between treatments
and plant types (t tests, ␣ ⫽ 0.05) are indicated in the text
and by asterisks in the corresponding figure or table.
RESULTS AND DISCUSSION
The ATP sulfurylase overexpressing (APS) Indian
mustard transgenics were shown previously to have en-
hanced rates of sulfate and selenate reduction and to
contain higher levels of total thiols, S, and Se (Pilon-
Smits et al., 1999). The objective of this study was to
explore the effects of ATP sulfurylase overexpression
on tolerance and accumulation of other metals, both
oxyanions and cations, reasoning that oxyanions may
react with the ATP sulfurylase enzyme directly, and
cations may be bound by thiols.
3
Fig. 1. Tolerance of wild-type (WT) and APS (i.e., overexpression
of ATP sulfurylase) transgenic Indian mustard seedlings to 12
metal(loid)s, supplied at concentrations as indicated in Table 1.
Tolerance is expressed as the ratio of root length in the presence
of metals relative to root length on control medium. Values shown
are the mean and standard error of the mean of 36 seedlings each.
Significant differences between APS and WT seedlings (␣ ⫽ 0.05)
are indicated by asterisks.
f
oo
Seedling Metal Tolerance and Accumulation
No significant differences in growth were observed
between APS and wild-type (WT) seedlings grown on
Pr
control agar medium without added metals. In the pres-
ence of metals there were some significant differences.
Judged from root growth inhibition, the APS seedlings
were significantly more tolerant than WT to As(III),
ed
As(V), Cd, Cu, Hg, and Zn, with the largest difference
on Cd where APS relative root growth was more than
double that of WT (Fig. 1). The APS seedlings were
less tolerant than WT to Mo and V, with the greatest
difference on Mo where relative APS root growth was
about one-half that of WT (Fig. 1). No significant differ-
ences in tolerance were found between APS and WT
plants for Cr, Mn, Ni, Pb, and W.
The enhanced tolerance of the APS plants to As(III),
As(V), Cd, Cu, Hg, and Zn could be due to their en-
hanced thiol levels. These elements were all shown to
be bound by phytochelatins (Maitani et al., 1996; Cob-
bett and Goldsbrough, 2000; Pickering et al., 2000;
Schmöger et al., 2000). Binding of excess amounts of
these metals by the thiol-containing peptides glutathi-
one and phytochelatins may facilitate their vacuolar se-
questration and detoxification, leading to enhanced tol-
erance.
The reduced tolerance of the APS seedlings to Mo
and V may be because ATP sulfurylase can react directly
with these oxyanions. Wilson and Bandurski (1958) re-
ported that ATP sulfurylase can use molybdate, tung-
state, chromate, and selenate as a substrate, but only
the reactions with sulfate and selenate resulted in stable
end products. If the products of these reactions are
unstable, this will lead to a loss of energy due to futile
cycling of ATP to ADP and back. Also, competition
with other oxyanions may lead to lower levels of the
normal end products of sulfate assimilation. Together,
this may explain the lower tolerance of the APS seed-
lings to Mo and V. The APS seedlings did not show
reduced tolerance to two other oxyanions tested (Cr
and W), however. These differences in oxyanion toler-
4 J. ENVIRON. QUAL., VOL. 33, JANUARY–FEBRUARY 2004
Fig. 2. Shoot metal concentrations in seedlings of wild-type (WT) and
APS (i.e., overexpression of ATP sulfurylase) transgenic Indian
mustard supplied with six metals, at concentrations as listed in
Table 1. Values shown represent the mean and standard error of
the mean of five samples consisting of six or seven seedlings each.
All metals shown here were present at significantly different con-
centrations (␣ ⫽ 0.05) between WT and APS seedlings. Additional
results from metals that were accumulated to a similar extent by
both plant types are shown in Table 2.
Table 2. Shoot metal concentrations in wild type (WT) and APS
(i.e., overexpression of ATP sulfurylase) seedlings after 7 d of
growth at the concentrations shown in Table 1.†
Treatment WT
re
mg kg⫺1 dry wt.
Cd 553 ⫾ 61
Cu 72 ⫾ 4
or
Mn 3581 ⫾ 141
Ni 217 ⫾ 8
W 317 ⫾ 45
Zn 1284 ⫾ 83
nc
† Values are the mean and standard error of the mean of five samples
consisting of six or seven seedlings each. The shoot concentrations of
the metals shown here were not significantly different between WT and
APS seedlings. Additional results from metals that were accumulated
U
to significantly different extents by the two plant types are shown in Fig. 2.
ance may be related to the different tissue concentra-
tions of these elements (see below).
The APS seedlings had significantly higher shoot con-
centrations than WT of As(III), As(V), Hg, Mo, Pb,
and V, with more than double the WT levels for both
forms of As and almost threefold higher Hg levels
(Fig. 2). The APS seedlings had somewhat (approxi-
mately 10%) lower Cr levels in their shoots than WT,
and there were no significant differences in shoot metal
concentrations between APS and WT when treated with
Cd, Cu, Mn, Ni, W, or Zn (Table 2).
The higher shoot levels of As and Hg in the APS
seedlings may be the result of their higher tolerance to
these elements, leading to better overall health, transpi-
ration, and root–shoot translocation. No differences in
shoot concentrations of other thiol-bound metals (Cd,
f
oo
Fig. 3. Shoot metal concentrations in wild-type (WT) and APS (i.e.,
overexpression of ATP sulfurylase) transgenic Indian mustard
plants supplied with six metals at the concentrations listed in
Table 1. Values shown represent the mean and standard error of
Pr
the mean of 10 plants, each sampled once. All metals shown here
were present at significantly different concentrations (␣ ⫽ 0.05)
between WT and APS plants. Additional results from metals that
were accumulated to a similar extent by both plant types are shown
in Table 3.
ed
Cu, and Zn) were found, however; perhaps these metals
are retained more in the root.
ct
The higher levels of Mo and V could be due to upregu-
lation of the sulfate uptake system, if these oxyanions
APS8
can make use of sulfate permease transporter proteins,
as suggested by certain studies (Leggett and Epstein,
495 ⫾ 72 1956; Marschner, 1995; Leustek, 1996). Upregulation of
73 ⫾ 4
3220 ⫾ 177 sulfate permease may be a reaction to overexpression
230 ⫾ 7 of ATP sulfurylase, and may also explain the enhanced
320 ⫾ 30
1361 ⫾ 68 levels of S and Se observed earlier in APS plants (Pilon-
Smits et al., 1999). The higher levels of Mo and V inside
the APS plants offer an additional explanation for their
lower tolerance to these elements. The APS seedlings
did not show enhanced accumulation of the other oxya-
nions Cr and W, possibly explaining why they showed
the same tolerance as wild type to these elements at the
seedling level.
Mature Plant Metal Tolerance and Accumulation
There were no significant differences in metal toler-
ance between APS and WT mature plants, as judged
from fresh weight gain when healthy mature plants were
exposed for 14 d to the 12 individual metals in a nutrient
film setup (results not shown).
The APS transgenics, however, did show enhanced
accumulation of a range of metals, both cations and
anions. The APS mature plants contained significantly
higher shoot concentrations than WT for Cd, Cr, Cu,
Mo, V, and W, with the Cd and V concentrations being
almost double and triple those of WT, respectively
(Fig. 3). The APS and WT plants showed no differences
 WANGELINE ET AL.: OVEREXPRESSION OF ATP SULFURYLASE IN INDIAN MUSTARD 5

Table 3. Shoot metal concentrations in mature plants after 14 d stimulate enhanced Fe uptake. The S for molybdenum
of treatment at the concentrations shown in Table 1.†
cofactor (MoCo) is also derived from cysteine (Amrani
Treatment WT APS8 et al., 2000), and enhanced availability of S may also
mg kg⫺1 dry wt. stimulate Mo uptake.
As 212 ⫾ 43 228 ⫾ 52 Not only were shoot S levels approximately 1.5-fold
Hg 34 ⫾ 17 17 ⫾ 6
Mn 3124 ⫾ 181 3324 ⫾ 234
higher under control conditions in APS plants, but vari-
Ni 35 ⫾ 3 35 ⫾ 2 ous metal treatments further enhanced this difference
Pb 47 ⫾ 13 27 ⫾ 5 between APS and WT. Treatment with As, Pb, and V,
Zn 515 ⫾ 22 547 ⫾ 30
for instance, resulted in a modest increase in WT S
† Values are the mean and standard error of the mean of 10 plants, each levels, but in a two- to threefold increase in APS S
sampled once. The shoot concentrations of the metals shown here were
not significantly different between wild type (WT) and APS (i.e., overex- levels. Treatment with Cr and Ni even resulted in a
pression of ATP sulfurylase) plants. Additional results from metals that decrease in WT S levels but a twofold increase in APS
were accumulated to significantly different extents by the two plant types
are shown in Fig. 3. S concentrations. Enzymes involved in sulfate uptake
and assimilation are known to be upregulated in re-
Table 4. Shoot concentrations of the essential elements Fe, Mo, and S in mature plants after 14 d of treatment with various metal(loid)s
at the concentrations shown in Table 1.
Fe Mo S
Treatment WT APS8 Comparison† WT APS8 Comparison† WT APS8 Comparison†

f
mg kg⫺1 dry wt. % mg kg⫺1 dry wt. % mg kg⫺1 dry wt. %

oo
Control 53 48 ⫺9 0.97 1.60 ⫹65* 7 491 10 755 ⫹44
As(III) 72 88 ⫹22* 1.16 1.71 ⫹47* 7 784 17 750 ⫹128*
Cd 57 49 ⫺14 3.4 9.38 ⫹176* 2 202 3 566 ⫹62*
Cr 77 47 ⫺39 0.49 1.65 ⫹237* 6 428 20 269 ⫹215*
Cu 25 46 ⫹84* 1.33 2.79 ⫹110* 2 209 4 368 ⫹98*

Pr
Hg 39 40 ⫹2 1.05 2.56 ⫹144* 2 496 4 894 ⫹96*
Mn 37 65 ⫹76* 0.71 3.94 ⫹455* 9 274 31 391 ⫹238*
Mo 38 37 ⫺3 2 784 3 917 ⫹41 1 328 1 987 ⫹50*
Ni 87 151 ⫹74* 0.72 2.37 ⫹229* 6 067 21 007 ⫹246*
Pb 48 66 ⫹38 1.94 3.47 ⫹79* 8 773 31 550 ⫹260*
ed
V 54 197 ⫹265* 2.56 2.71 ⫹6 11 953 27 284 ⫹128*
W 47 49 ⫹4 2.44 1.64 ⫺33 1 002 1 762 ⫹76*
Zn 26 41 ⫹58* 1.15 2.76 ⫹140* 2 593 4 658 ⫹80*
Average ⫹47 ⫹135 ⫹139
ct

† Comparison of APS8 (i.e., overexpression of ATP sulfurylase) relative to wild type (WT). Asterisks indicate a statistically significant difference between
APS8 and WT (␣ ⫽ 0.05).

in shoot metal concentration when supplied with sponse to metal stress to provide cysteine for the produc-
re

As(III), Hg, Mn, Ni, Pb, and Zn (Table 3). tion of metal-binding thiols (Chen and Leustek, 1995;
Of the metals or metalloids accumulated to higher Logan et al., 1996). The presence of extra ATP sulfu-
or

levels in shoots of APS transgenics compared with WT, rylase activity appears to somehow stimulate this effect,
four were supplied as oxyanions (Cr, Mo, V, and W) perhaps by further enhancing sulfate permease levels.
and their enhanced uptake may be due to upregulation Similar but less pronounced differences were ob-
nc

of sulfate transporter systems, as hypothesized above. served for Mo and Fe (Table 4). The shoot Mg levels
The other two, Cd and Cu, can be complexed by thiols; were also somewhat enhanced in APS plants relative
perhaps more efficient binding by thiols can somehow to WT (⫹20% on average; p ⬍ 0.05 for nine treatments);
U

stimulate their uptake. The same effect was not ob-
served, however, for other thiol-bound elements such
as As and Hg.
To investigate the effect of ATP sulfurylase overex-
pression on plant levels of other, essential macro- and
micronutrient shoot concentrations of Cu, Fe, Mg, Mn,
Mo, S, and Zn were determined by ICP–AES for all
mature plant treatments. Interestingly, the APS plants
had higher levels of the essential elements Fe, Mo, and
S in the majority of treatments, with on average 1.5-
fold higher Fe levels and more than 2-fold higher Mo
and S levels (Table 4). The higher Mo and S levels may
be the result of upregulation of sulfate transporters as
a result of ATP sulfurylase overexpression, if molybdate
can be taken up by sulfate permease. The reason for
the higher Fe levels is not clear. Some plant Fe is com-
plexed with S in iron–sulfur clusters, which obtain their
S from cysteine (Amrani et al., 2000; Mihara et al.,
2002). Higher availability of S for Fe–S clusters may
there were no differences in Cu, Mn, or Zn levels (results
not shown).
Overall Effect of ATP Sulfurylase
Overexpression on Metal Tolerance
and Accumulation
There were similarities but also many differences be-
tween seedlings and mature plants with respect to metal
tolerance and accumulation. The differences in results
obtained with seedlings and mature plants may in part
be explained by development-related differences in
plant metabolism (e.g., differences in endogenous ATP
sulfurylase activity). Also, the differences observed be-
tween seedlings and mature plants may in part be due
to the experimental setup: the seedlings were exposed
to the metals continuously from seed, while the mature
plants were only exposed during the last 2 wk of their
8-wk life. In any case, the different results obtained from
6 J. ENVIRON. QUAL., VOL. 33, JANUARY–FEBRUARY 2004
seedlings and mature plants illustrate that one should
be careful when drawing conclusions about mature plant
metal tolerance and accumulation from experiments
with seedlings, and that tolerance by mature plants to
acute metal stress may involve different mechanisms
than continuous metal tolerance starting from germi-
nation.
In spite of the differences in results obtained from
seedlings and mature plants, some general trends
emerge with respect to the effect of ATP sulfurylase
overexpression on plant tolerance and accumulation of
oxyanions and cations. The APS plants showed a ten-
dency to accumulate enhanced shoot levels of oxyani-
ons. Shoot Mo and V levels were enhanced in both APS
seedlings and mature plants, Cr and W were higher in
mature APS plants, and As levels were enhanced in
APS seedlings. Tolerance to oxyanions tended to be
reduced or unaffected, with the exception of the thiol-
bound oxyanion As, to which APS seedlings had higher
tolerance. The APS plants contained enhanced shoot
levels of certain cations as well, especially thiol-bound
cations: Hg and Pb in seedlings and Cd and Cu in mature
plants. Seedling tolerance to cations tended to be en-
hanced, especially for known thiol-bound cations. Possi-
ble explanations for these phenomena have already
been discussed above.
Does Overexpression of ATP Sulfurylase Show
ed
Promise for Metal Phytoremediation?
The APS transgenics tended to accumulate metals to
higher shoot concentrations than wild type: of the 12
ct
elements examined, the APS plants contained up to 2.5-
fold higher levels of As, Cd, Cr, Cu, Hg, Mo, Pb, V,
re
and W in at least one growth stage. A higher shoot
metal concentration is a favorable property for phytoex-
traction. On the other hand, the APS plants tended
or
to be less tolerant to oxyanions. Whether decreased
tolerance would be a factor in a field situation remains
to be seen. Promising in this respect, in a study where
nc
plants were grown from seed on metal-contaminated
environmental soil, APS transgenics removed more Cd,
Cr, Cu, Mn, Pb, and Zn from the soil in comparison
U
with wild-type Indian mustard without any differences
in biomass production (Bennett et al., 2002). Thus, at
less extreme metal concentrations, metal tolerance does
not appear to negatively affect the use of APS plants
for metal extraction. The finding that APS transgenics
show enhanced accumulation of a variety of metals and
metalloids suggests that overexpression of ATP sulfu-
rylase is a promising strategy for the creation of plants
with enhanced metal phytoremediation capacity, espe-
cially in view of the fact that most metal-polluted sites
contain mixtures of metals. Of course any field use of
transgenic plants for phytoremediation should be pre-
ceded and accompanied by a thorough risk assess-
ment study.
ACKNOWLEDGMENTS
This work was supported by USEPA Research Grant
G8A11586 awarded to E.A.H. Pilon-Smits and N. Terry, and
U.S. National Science Foundation Grant MCB-9982432
awarded to E.A.H. Pilon-Smits, including a Research Experi-
ence for Undergraduates supplement for S.D. Lindblom. M.
Pilon is supported by NSF Grant MCB-0091163.
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