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Research Paper 1

Evaluation of the Metal Phytoextraction Potential of Crop Legumes.

Regulation of the Expression of O-Acetylserine (thiol)lyase
under Metal Stress
E. Pajuelo1, 2, J. A. Carrasco2, L. C. Romero3, M. A. Chamber2, and C. Gotor3
1
Present address: Departamento de Microbiologa y Parasitologa, Facultad de Farmacia, Universidad de Sevilla, Profesor Garca Gonzlez, 2,
41012 Sevilla, Spain
2
CIFA Las Torres-Tomejil, IFAPA, Junta de Andaluca, Carretera Sevilla-San Jos de la Rinconada, km. 12, 41200 Alcal del Ro, Sevilla, Spain
3
Instituto de Bioqumica Vegetal y Fotosntesis, Centro de Investigaciones Cientficas Isla de la Cartuja, CSIC and Universidad de Sevilla,
Avda. Amrico Vespucio, 49, 41092 Sevilla, Spain

Received: March 8, 2007; Accepted: May 22, 2007

Abstract: The metal phytoextraction potential of three legumes OAS: O-acetylserine

belonging to different genera has been studied under green-
house conditions. Legumes accumulate As and metals mainly in
roots, although translocation to shoot is observed. Alfalfa did
accumulate the highest concentrations of As and metals in
shoots and aerial biomass was less affected by the toxic ele-
ments, indicating its good behaviour in phytoextraction. Clover
accumulated less metal, but showed larger biomass. EDTA addi-
tion enhanced Pb phytoextraction up to levels similar to those
described for plants proposed in phytoremediation. The regula-
tion of O-acetylserine (thiol)lyase from legumes under metal
stress has been analysed to test the possibility of establishing a
possible correlation between the expression of OASTL in the
presence of the metals and the metal accumulation in legume
plant tissues. Cd and Pb(EDTA) produce the strongest increases
of OASTL activity, with the higher enhancement seen in roots, in
parallel with the higher metal accumulation. Arsenic produced
an increase of root enzyme activity, whereas Cu produced a de-
crease, mainly in shoots. Western blots using antibodies against
an A. thaliana cytosolic OAS-TL recognised up to five protein
bands in crude extracts from Lotus and clover. A low molecular
weight isoform of 32 kDa was induced in the presence of Cd
and Pb. A partial RT-PCR sequence form clover has been ob-
tained, showing 86 – 97 % identity with other described OASTLs.
The PCR fragment has been used to analyse OASTL mRNA levels
of legumes under metal stress. OASTL transcripts were increased
by As, Cd, and Pb, especially in roots, where metal accumulation
was maximal, while Cu produced a decrease in the transcript
levels.
Key words: Arsenic, cysteine biosynthesis, heavy metals, Lotus
japonicus, Medicago sativa, Trifolium subterraneum.
Abbreviations:
DTT: dithiothreitol
EDTA: ethylene-diamino tetraacetic acid
ICP-OES: induced coupled plasma optical spectrometry
kDa: kilodalton
Plant Biol. 9 (2007): 1 – 10
 Georg Thieme Verlag KG Stuttgart · New York
DOI 10.1055/s-2007-965439
ISSN 1435-8603
OASTL: O-acetylserine (thiol)lyase
PCs: phytochelatins
PLP: pyridoxal phosphate
PMSF: phenylmethylsulfonyl fluoride
PVDF: polyvinyldifluoride
SAT: serine acetyltransferase
TCA: trichloroacetic acid
Introduction
Contamination of soils and waters by heavy metals as a conse-
quence of mining activity, mine tailing accidents, or naturally
occurring geochemical processes, is a serious environmental
problem. Unlike contamination by compounds such as organ-
ics, metals cannot be degraded, but can only be removed and
stored in safe places, or, alternatively, stabilised in non-avail-
able forms in the soil. Current engineering-based technologies
used to clean up soils – such as the removal of contaminated
topsoil for storage in landfills – are very costly and dramatical-
ly disturb the landscape. Since both microorganisms and
plants have developed the ability to tolerate and/or detoxify
metals, phytoremediation is used as a cost-effective, environ-
mentally friendly, alternative technology for soil reclamation
(Pilon-Smits, 2005; McIntyre, 2003). Certain plant species –
known as metal hyperaccumulators – have the ability to live
in soils extremely polluted by certain heavy metals. Hyperac-
cumulators can extract elements from the soil and concentrate
them in the easily harvested plant shoots at very high concen-
trations. About 400 plant species in 45 different families are
known to hyperaccumulate metals (Ma et al., 2001; Baker et
al., 2000). There are biodiversity prospecting efforts around
the world looking for hyperaccumulator plants in highly pol-
luted soils (Prasad and Freitas, 2003). Unfortunately, the use
of hyperaccumulating plants is limited by their small biomass,
slow growth rates, and unknown agronomic potential. This
has led scientists to propose the use of crop plants producing
high biomass in phytoremediation, and some plants, such as
several Brassica species, Nicotiana glauca, maize, sunflower,
and sorghum, have been used in phytoremediation experi-
ments (Bricker et al., 2004; Del Ro et al., 2004; Lombi et al.,
2001; Murillo et al., 1999).

2 Plant Biology 9 (2007)
The mechanism of metal resistance in plants is still not com-
pletely understood, although it seems clear that different
mechanisms operate in hyperaccumulators and non-hyperac-
cumulators (Callahan et al., 2006; Wang et al., 2002; Pence et
al., 2000). Non-hyperaccumulators can synthesise two types of
cysteine-rich peptides, phytochelatins and methallothioneins,
together with other metal-binding molecules, such as histi-
dine, nicotianamine, and organic acids like citrate. Metals can
also be accumulated in inert cell structures such as cell walls
(Pilon-Smits, 2005). Some metals, e.g., Zn, Cu, or Ni, are mainly
detoxified via metallothioneins and histidine (Persans et al.,
1999; Evans et al., 1992). The other peptides, phytochelatins,
are g-glutamylcysteine oligomers, the synthesis of which is
rapidly induced from glutathione in the presence of arsenic
and metals such as Cd, Hg, Ag, and Pb, in order to chelate met-
als throughout the thiol moiety of the cysteine, and sequester
them in the vacuoles (Clemens, 2006; Pilon-Smits, 2005). Cys-
teine is synthesised from serine and sulfide in a two-step reac-
tion catalysed by the cysteine synthase complex (Wirtzn and
Hell, 2006). In the first step, serine acetyltransferase transfers
an acetyl group to serine, producing O-acetylserine. In the sec-
ond step, the O-acetylserine (thiol)lyase (OASTL) catalyses cys-
teine biosynthesis from O-acetylserine and sulfide. Previous
work has demonstrated the regulation of OASTL from Arabi-
dopsis thaliana by heavy metals such as Cd (Domnguez-Sols
et al., 2004, 2001). Cd induces OASTL activity in the leaf lam-
ina and also in the stem and root cortex (Domnguez-Sols et
al., 2001). In the leaf, Cd is mainly accumulated in the tri-
chomes (Ager et al., 2002), which act as a sink during detoxi-
fication processes (Gutirrez-Alcal et al., 2000). Furthermore,
genetic manipulation of the enzymes of the phytochelatin
biosynthesis pathway has been shown to increase plant toler-
ance to heavy metals or metal accumulation. Overexpression
of OASTL and of enzymes from the phytochelatin biosynthe-
sis pathway, phytochelatin synthase, glutathione synthetase,
or g-glutamylcysteine synthetase, led to an increase in Cd re-
sistance and/or accumulation in Arabidopsis thaliana and other
high biomass-producing plants (Domnguez-Sols et al., 2004;
Sirko et al., 2004; Gisbert et al., 2003; Song et al., 2003; Zhu et
al., 1999 a, b).
Legume plants have been widely used in soil regeneration due
to their ability to grow in poor soils. Legumes establish a sym-
biotic interaction with Rhizobium, allowing them to grow in
the absence of any external nitrogen supply. They are widely
used in soil improvement in agronomic practices such as crop
rotation, since they improve the growth of other plants due to
soil nitrogen enrichment (Graham and Vance, 2003). Among
legumes, many species are known to tolerate metals and grow
in soils with high metal concentrations. These legumes include
Anthyllis, Cytisus, Lotus, Genista, Ononis, Ornithopus, several
Trifolium species, Vicia, etc. (Prasad and Freitas, 2003). Legume
plants have been recently proposed as new tool in soil recla-
mation (Carrasco et al., 2005; Pastor et al., 2003; Sriprang et
al., 2002).
In this study, we conducted a series of pot experiments in or-
der to assess the phytoextraction capacity of the model legume
Lotus japonicus and two crop legume plants (alfalfa and clo-
ver), and the effect of As and metals on plant biomass and tox-
icity symptoms. The effect of the addition of EDTA in improv-
ing Pb uptake and translocation to the shoot in legume plants
has also been studied. Furthermore, in an attempt to find a cor-
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E. Pajuelo et al.
relation between the expression of OASTL in the presence of
the metals and metal accumulation in legumes, we analysed
the regulation of OASTL in the model legume plant Lotus japo-
nicus and crop legumes growing in the presence of As and
three heavy metals (Cd, Cu, and Pb). Enzyme activity and pro-
tein levels have been determined. The levels of OASTL mRNA in
roots and leaves under metal stress have been analysed using a
partial Trifolium subterraneum OASTL as probe (this work).
Materials and Methods
Plant growth conditions
Three legume species were used in this study: Medicago sativa,
Trifolium subterraneum, and the model legume Lotus japonicus.
Seeds from Medicago sativa were surface-sterilised with 70%
ethanol for 20 min and washed repeatedly with distilled wa-
ter. Other legume seeds with harder seed coats needed treat-
ment with concentrated sulfuric acid for 5 min, and washing
five times with distilled water. After pre-germination for 24 h
on wet filter paper in petri dishes at 20 8C, imbibed seeds
were put in seed trays filled with 50 % perlite : 50 % vermiculite.
Plants were cultivated in a plant growth chamber at 22 8C/15 8C
day/night temperatures and 16 h/8 h day/night photoperiod.
Every three days the plants were watered with plant growth
solution (Serrano and Chamber, 1990) plus 5 mM potassium
nitrate, containing either 2 mg l–1 arsenic (26.7 M) (in the
form of sodium arsenite), 2 mg l–1 cadmium (17.8 M) (cad-
mium chloride), 50 mg l–1 copper (0.78 mM) (copper sulfate),
or 500 mg l–1 lead (2.4 mM) (lead nitrate plus 5 mM EDTA to
avoid precipitation of lead). Plants watered without metal ad-
dition were used as a control. After 2 months, the aerial part of
plants and roots were harvested separately, weighed for bio-
mass determination, and stored at – 80 8C until use.
Determination of metal accumulation in plants
Arsenic or metal accumulation was determined according to
Murillo et al. (1999). Samples of roots (approx. 0.5 g) or aerial
parts (stems plus leaves, approx. 1 g) of the abovementioned
plants were dried for 48 h at 60 8C and weighed for dry matter
determination. Acid digestion of organic matter was per-
formed in 20 ml of HClO4 : HNO3 7: 1 for 24 h at 60 8C, until
the final volume was reduced to 1 ml. Double-distilled water
(milli-Q) was added up to 100 ml and the resulting solution
was filtered through a 0.45-mm filter. As or heavy metals were
determined in this solution by ICP-OES. Metal concentration
was related to dry matter (mg kg–1 dry weight). Data are
means of two independent determinations in samples ob-
tained by mixing material from 10 plants in each replicate.
Determination of OASTL activity in crude extracts
OASTL activity in crude extracts from roots and shoots was de-
termined according to Barroso et al. (1995) in the abovemen-
tioned plant samples. Samples of 0.5 g roots or 1.0 g shoots
were triturated with a mortar and pestle in liquid nitrogen
and homogenised in five volumes of extraction buffer (50 mM
potassium phosphate pH 7.5 containing 1 mM EDTA, 1 mM
DTT, 0.5 mM PMSF, and 10 M pyridoxal 5¢-phosphate). After
centrifugation, OASTL activity was assayed in the cleared su-
pernatant. The reaction mixture contained 50 mM potassium
phosphate buffer pH 7.5, 20 mM OAS, 10 mM Na2S, and 50 –

Legume Phytoextraction Potentia
100 l of crude extract. The reaction mixture was incubated
at 30 8C for 10 min and stopped by addition of 60 l of 20 %
TCA. Cysteine was determined by measuring optical density
at 506 nm after the formation of a complex with nynhidrin.
Total protein in crude extracts was determined by the method
of Bradford (1976). Data are means of three independent deter-
minations.
Western blot analysis
Samples of the previously obtained crude extracts from shoots
(containing 30 g protein) or roots (containing 15 g protein)
were electrophoresed on 12% acrylamide gels and transferred
to PVDF membranes using the NovaBlot system (Pharmacia)
according to instructions from the manufacturer. Western blot
was performed using polyclonal antibodies raised against the
cytosolic OASTL from Arabidopsis thaliana, generously provid-
ed by R. Hell (Heidelberg Institute of Plant Sciences, Germany),
and secondary antibodies of conjugated alkaline phosphatase
(Sigma).
RT-PCR amplification of an OASTL fragment from
Trifolium subterraneum
Degenerated PCR primers were designed based on the se-
quence of the major cytoslic OASTL from A. thaliana (OAS-A1,
Barroso et al. [1995], accession number: X84097.1) and the
OASTLs from legumes described in the database (AF452451.1
from Glycine max, AJ006024.1 from Cicer arietinum, and
AC135320.6 from Medicago truncatula). The mRNA from le-
gumes was isolated using the RNAeasy Plant minikit (Qiagen),
following the instructions provided. The RT-PCR reaction was
done using the Superscript II RT (Invitrogen) according to
the recommendations included. The degenerated primers used
for RT-PCR amplification were: forward: 5¢-TTG GAT TAG CCT
TYA TGG CAG-3¢, and reverse: 5¢-CWA TCT KAA WVG CAG
CGG C-3¢. The RT-PCR product of Trifolium subterraneum was
removed from the gel and purified with the GFX PCR DNA
and Gel Band Purification kit (Amersham), cloned in pGEM-
Teasy (Promega) and sequenced.
Northern blot analysis
Total RNA from leaves or roots of the abovementioned plants
grown on metals or under control conditions was obtained us-
ing the method previously described by Gotor et al. (1997).
Samples of 10 g of total RNA were electrophoresed on formal-
dehyde gels and blotted to membranes of Z-probe() Blotting
(Biorad) by capillary transfer. RNA was fixed at 80 8C for 1 h. Fil-
ters were prehybridised for 2 h at 65 8C in 0.5 M sodium phos-
phate buffer pH 7.2 containing 1 mM EDTA and 7% SDS. La-
belled probes were added and hybridisation continued over-
night using the PCR fragment of Trifolium subterraneum OASTL
(this work) as probe. Membranes were washed twice in 40 mM
sodium phosphate buffer pH 7.2 containing 1 mM EDTA and
4% SDS at 55 8C for 15 min each, plus two washes in 40 mM so-
dium phosphate buffer containing 1 mM EDTA and 1% SDS at
55 8C for 15 min each. The filter was placed in front of a Fujifilm
Imaging Plate BAS-MS 2040 for 24 h at room temperature and
signals were detected and analysed in a BioScience “Cyclone
Storage Phosphor System”. The same filters were washed in
boiling 0.5 % SDS in order to remove all radioactivity, and re-
hybridised with the maize 17S DNA as a control for total RNA
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Plant Biology 9 (2007) 3
quantity. Images were analysed using the software provided
by the manufacturer, and normalised for the signal of the 18S
RNA band.
Results
Evaluation of phytoextraction capacity in model and
crop legumes: a pot experiment
As the final aim of our work is studying the possibilities of us-
ing legumes in phytoextraction, we wanted to determine the
maximum amount of As and heavy metals that legume plants
can tolerate and accumulate. The concentrations of As and
heavy metals used in this experiment were the highest that
these plants could tolerate with reasonable seed germination
and growth. Although the concentrations of metals are appar-
ently high, they were similar to the level previously reported
by Carrasco et al. (2005) for heavy metal-contaminated soils
where native legume plants were found, such as in a contami-
nated area affected by a mine toxic spill (Grimalt et al., 1999).
As an initial step to evaluate the potential of legume plants in
metal phytoextraction, the effect of the presence of As and
heavy metals on growth of legumes was evaluated, since total
phytoextraction is the product of metal accumulation in the
aboveground part of the plant by the total shoot biomass. The
effect of the presence of As and heavy metals (Cd, Cu, and Pb)
on biomass was measured for a model (Lotus japonicus) and
two crop legumes (alfalfa and clover) in a long-term pot ex-
periment conducted in the greenhouse. Data on legume bio-
mass grown in the presence of toxic elements are given in
Table 1. In the absence of any toxic element, clover had a higher
shoot biomass, followed by alfalfa. Also, the ratio of the bio-
mass of shoots to roots is highest in clover, a very important
characteristic for phytoextraction, since shoots accumulating
metals are removed in the phytoremediation technique. In
general terms, the presence of As or any of the metals assayed
produced a decrease in shoot biomass of legume plants. Cu
was the metal that had the largest effect on legume biomass
of both shoot and root tissues at the concentrations used in
the experiment. Cu concentration was very high but resembled
natural concentrations in some contaminated areas where le-
gumes can actually grow. The decrease in shoot biomass in the
presence of Cu was between 40 and 75%. Also, the relationship
between the biomass of shoots and roots decreased, with min-
imum values found in the presence of Cu, indicating that long-
term treatment with Cu has a larger effect on shoot growth
than root biomass. In fact, plants grown in the presence of
50 ppm Cu had very small shoots, with dark green, small trifo-
liate leaves and red stems. These toxicity symptoms, which
were apparent in all legumes tested, were even stronger in Lo-
tus japonicus plants (not shown). The presence of As, Cd, or Pb
also produced a reduction in shoot biomass of 20 to 40 %, on
average. However, the growth of alfalfa shoots in the presence
of Cu, Cd, or Pb did not show a reduction, and the reduction in
shoot biomass was least in alfalfa (66 %) compared to the other
legumes. These results probably indicate a better tolerance of
alfalfa to these toxic elements. The combined presence of Pb +
EDTA produced an increase in the shoot to root biomass ratio.
This effect was remarkable in alfalfa and clover plants, which
had very big shoots, compared to a very small root system. This
increase in shoot biomass could be an advantage in phyto-
extraction.
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4 Plant Biology 9 (2007) E. Pajuelo et al.

Table 1 Effect of arsenic and heavy metals on the growth of legume plants. Plants were grown in the presence of either As (26.7 M), Cd
(17.8 M), Cu (0.78 mM), or Pb (2.4 mM), or in the absence of any toxic element (none). Shoot and root biomass were determined after 2 months
growth. Data show means and standard deviations corresponding to four independent replicates. Percentage with regard to biomass in the ab-
sence of toxic elements is given in parentheses

Legume species Element FW per plant (mg) Shoot/Root biomass

Shoot Root

Lotus japonicus none 126 € 32 52 € 9 2.42

As 100 € 30 (79%) 40 € 11 (77%) 2.50
Cd 73 € 24 (58%) 47 € 9 (90%) 1.55
Cu 30 € 8 (24%) 23 € 5 (44%) 1.30
Pb (5 mM EDTA) 66 € 10 (52%) 27 € 7 (46%) 2.44

Medicago sativa none 269 € 70 176 € 32 1.53

As 180 € 115 (66%) 112 € 47 (67%) 1.59
Cd 276 € 77 (103%) 172 € 39 (98%) 1.61
Cu 172 € 39 (66%) 169 € 46 (96%) 1.03
Pb (5 mM EDTA) 285 € 10 (105%) 67 € 10 (37%) 4.29

Trifolium subterraneum none 781 € 100 181 € 65 4.32

As 633 € 122 (81 %) 183 € 45 (100%) 3.46
Cd 496 € 71 (63%) 182 € 42 (100%) 2.72
Cu 362 € 54 (46%) 94 € 29 (52%) 3.85
Pb (5 mM EDTA) 634 € 70 (81%) 100 € 30 (55%) 6.34

Table 2 Accumulation of arsenic and heavy metals in shoots and roots of legume plants. Arsenic and metal accumulation were determined in
shoot and root tissues of the same plant samples as in Table 1. Data are means of two independent determinations in which shoot or root tissues
from 10 plants were mixed (n.d., not determined)

Legume species Tissue Accumulation (mg/kg dry weight)

As Cd Cu Pb + 5 mM EDTA

Lotus japonicus shoots <1 26.5 € 3.5 18.5 € 3.7 572 € 232
roots 336 € 126 116 € 45 144 € 31 n.d.

Medicago sativa shoots 85 € 15.3 53 € 10.3 77 € 22.5 2 177 € 424

roots 1 131 € 147 677 € 136 988 € 229 868 € 179

Trifolium subterraneum shoots 12.6 € 4.8 29.5 € 2.7 52.5 € 7.9 2 180 € 344
roots 656 € 207 545 € 132 643 € 82 1 983 € 256

The phytoextraction capacity of legumes grown in the pres-
ence of As and heavy metals (Cd, Cu, and Pb) was evaluated in
the same plants in which final metal accumulation after 2-
month growth was measured. Table 2 shows data on metal
accumulation in the aboveground part of plants (stems plus
leaves) and in roots. Legume plants accumulated As and met-
als mainly in the root tissue, as expected for non-hyperaccu-
mulators. Arsenic and metal accumulation was higher in roots
of all legume plants, especially alfalfa, whereas Lotus japonicus
had the lowest accumulation, together with stronger toxicity
symptoms (not shown). However, significant translocation of
As and heavy metals to the aboveground part of legume
plants was also observed. Alfalfa had the highest concentra-
tion of metals in shoots, with a ratio of metal accumulation in
shoots to roots close to 0.1. Another good characteristic of met-
al accumulation in alfalfa is the lack of deep toxicity symptoms
and biomass reduction in the presence of Cd and Pb, which
could be an indication that this particular legume could toler-
ate higher metal concentrations. In contrast, Lotus japonicus
seems to accumulate low amounts of As in shoots (ratio of As
accumulation in shoots to roots of 0.01), behaving as an As ex-
cluder. With regard to Cd and Cu, Lotus accumulated lower
concentrations in shoots than alfalfa. Nevertheless, the phy-
toextraction (i.e., the ratio of shoot to root metal accumula-
tion) was 0.22 and 0.12, respectively, higher than in alfalfa.
Clover had intermediate concentrations of metals (As, Cd, and
Cu) in shoots. However, considering the biomass production
shown in Table 1, it could be concluded that clover is able to
phytoextract about the same amount of metals (Cu and Pb) as
alfalfa, since biomass production in the presence of metals is
two to three times higher than alfalfa. In all cases, the addi-
tion of EDTA increased both accumulation and translocation
of Pb into the aerial part of the plants. Pb accumulation under
these conditions is over 2000 ppm in shoots of alfalfa and clo-
ver, very similar to the levels described for other high-biomass
plants proposed in phytoremediation. The ratio for Pb accumu-
lation in the presence of EDTA reached up to 1.1 in clover and
up to 2.5 in alfalfa, indicating that, in global terms, alfalfa
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Legume Phytoextraction Potentia Plant Biology 9 (2007) 5

Table 3 Effect of As and heavy metals on OASTL activity in roots and leaves of legume plants. Plants were grown in the presence of either
As (26.7 M), Cd (17.8 M), Cu (0.78 mM), or Pb (2.4 mM), or in the absence of any toxic element (none). After 2 months, the aerial part of plants
and roots were harvested separately, crude extracts prepared and OASTL activity determined as described in materials and methods. Data are
means of three independent determinations € standard deviations. Percentages with regard to activity in the absence of toxic elements are given
in parentheses

Legume species Element OASTL activity in leaves OASTL activity in roots

(mU mg–1 protein) (mU mg–1 protein)

Lotus japonicus none 227 € 24.4 215 € 14.7

As 209 € 22.6 (92%) 238 € 16.2 (111 %)
Cd 241 € 17.3 (106%) 289 € 20.5 (134%)
Cu 202 € 21.3 (89%) 198 € 38.8 (92%)
Pb (5 mM EDTA) 297 € 14.2 (131%) 301 € 32.3 (140%)

Medicago sativa none 243 € 22.2 230 € 26.8

As 208 € 17.4 (85%) 350 € 35.2 (152%)
Cd 360 € 23.7 (148 %) 322 € 27.5 (140 %)
Cu 120 € 19.9 (49%) 210 € 20.2 (91%)
Pb (5 mM EDTA) 402 € 22.7 (165%) 400 € 33.7 (174%)

Trifolium subterraneum none 244 € 33.4 n.d.

As 220 € 24.5 (90%) n.d.
Cd 250 € 43.6 (102%) n.d.
Cu 156 € 26.8 (64%) n.d.
Pb (5 mM EDTA) 276 € 34.2 (113 %) n.d.

plants can phytoextract considerably high amounts of Pb un-
der these conditions, in terms of both the increase in shoot bio-
mass (Table 1) and Pb accumulation (Table 2).
Effect of the presence of As and heavy metals on OASTL
activity and protein in roots and shoots of legume plants
Since O-acetylserine (thiol)lyase has been proved to be one of
the enzymes highly modulated by the presence of heavy met-
als (Domnguez-Sols et al., 2001), the effect of the presence of
As and heavy metals on enzyme activity has been determined
in an attempt to find a correlation between the expression of
legume OASTL and metal accumulation. OASTL activity was
measured in crude extracts from shoots and roots of Lotus
japonicus and crop legumes grown in the presence of As and
heavy metals (Cd, Cu, and Pb) (Table 3). Cd and Pb produced
an increase in OASTL activity, in shoots but especially in roots
of legume plants, corresponding to higher metal accumulation
in the root tissue. In comparison with control plants, the activ-
ity increases ranged between 10 to 75%, depending on the
plant species and tissue. The highest increase was detected in
the presence of 500 ppm Pb + 5 mM EDTA (between 50 – 70%
in most cases), again in agreement with the very high levels
of Pb accumulation in the presence of EDTA. In contrast, the
presence of Cu (50 ppm) did not produce an increase in OASTL
activity, but produced a decrease in this activity of between 20
and 50%, compared to control plants. This effect was observed
mainly in shoots. The presence of As also produced an increase
in OASTL activity in alfalfa and Lotus, but only in roots, where
As accumulation is higher (Table 2).
In order to see if the increase in OASTL activity is correlated
to protein levels, a Western blot experiment was conducted,
using antibodies raised against the most abundant cytosolic
OASTL isoenzyme from Arabidopsis thaliana. These antibodies
recognised up to four different protein bands in Arabidopsis
crude extracts (used as control in Western blots), and up to five
different bands in legume plants, depending on the legume
species and plant tissue (Fig. 1). In Lotus japonicus, four OASTL
protein bands with molecular masses between 32 and 36 kDa
were detected (Fig. 1 A), whereas in Trifolium subterraneum
up to five different OASTL isoenzymes were detected by the
A. thaliana antibodies, with molecular masses between 32
and 36 kDa (Fig. 1 B). The intensity of protein bands did not
show large variations in the presence of metals. Nevertheless,
it is particularly interesting to find strong and specific en-
hancement of an OASTL isoform with a lower molecular mass
(32 kDa), particularly in the presence of Cd and Pb, and espe-
cially in clover plants (labelled with a small arrow in Fig. 1 B).
This isoenzyme was also induced by Cd in roots of clover.
Effect of the presence of As and heavy metals on the OASTL
mRNA levels in legume plants
To further study the expression of OASTL from legumes in re-
sponse to As and metal stress, we performed Northern blot
analysis. In a first experiment, the major cytosolic OASTL from
Arabidopsis thaliana (OAS-A1, Barroso et al., 1995) was used as
the probe to analyse the metal regulation of legume OASTL on
Northern blots. However, in our conditions, we could not see
clear hybridisation bands between the Arabidopsis probe and
legume mRNAs. Therefore, to obtain a homologous probe, a
fragment of the OASTL from legumes was amplified by PCR.
Degenerated primers based on the sequence of legume OASTLs
found in the database were designed and used for amplifica-
tion of an OASTL fragment from legumes studied in a RT-PCR
reaction using mRNA. The primers only allowed the amplifica-

6 Plant Biology 9 (2007)
tion of a 585-bp DNA fragment in clover (not shown). No am-
plification bands could be obtained for alfalfa and Lotus using
these primers, either with mRNA or genomic DNA (not shown).
The deduced amino acid sequences of the OASTL fragment
from Trifolium subterraneum (GeneBank accession number
EF193210) is shown in Fig. 2. The partial sequence showed a
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Fig. 1 Western blot analysis of crude ex-
tracts form leaves and roots of (A) Lotus japo-
nicus and (B) Trifolium subterraneum, grown in
the presence of As and heavy metals. 30 g of
total protein from leaves or 15 g of total pro-
tein from roots were electrophoresed and
blotted to nylon membranes. OASTL isoen-
zymes were detected using antibodies raised
against A. thaliana major cytosolic OASTL.
Crude extract from leaves of Arabidopsis thali-
ana was also subjected to Western blot analy-
sis as control. Lanes C correspond to crude ex-
tracts from plants grown in control conditions
in the absence of any As or heavy metals. Ar-
rows show different OASTL isoforms.
Fig. 2 Sequence analysis of OASTL from Tri-
folium subterraneum. Partial sequence (585 nt)
of clover OASTL is shown compared to the en-
zyme from other legumes (chickpea and soy-
bean) and A. thaliana. Residues involved in
enzyme activity and/or ligands binding are
shown in boxes.
94 – 97% identity with the same enzyme from other legumes
(chickpea, soybean), a slightly lower identity with M. trunca-
tula OASTL (91 – 92 %), and 83 – 84 % identity with the A. thali-
ana protein. The OASTL belongs to the cysteine synthase/cys-
tathionine beta-synthase family, a class of pyridoxal-depen-
dent enzymes. Several conserved motifs are also present in

Legume Phytoextraction Potentia
the enzyme from clover, highlighted in Fig. 2. The motif
G(181)TGGT(185) is conserved in the enzyme from clover, and
is reported to participate in pyridoxal-phosphate binding, as
well as Ser (269), which is also conserved in our sequence. On
the other hand, the sequence KPGPHK, also present in the clo-
ver partial sequence, was identified as a SAT1 binding site in
the A. thaliana cysteine synthase complex. Recently, the reso-
lution of the crystal structure of several OASTLs and mutagen-
esis studies have shown that Thr(74) and Ser(75) are involved
in the incorporation of sulfur to O-acetylserine, whereas
Gln(147) and Asn(77) have been proposed to interact with O-
acetylserine (Bonner et al., 2005). Gln(147) is also conserved
in the partial sequence of the enzyme from clover (Fig. 2).
The Trifolium subterraneum OASTL PCR fragment (585 bp) was
used as the probe to study the OASTL mRNA levels in the pres-
ence of As and heavy metals in legumes on Northern blot anal-
ysis. The results of hybridisation can be seen in Fig. 3. In Lotus
japonicus, several mRNA bands hybridised with the OASTL
probe (not shown). However, after filter washes with more
stringent conditions, a band of approx. 1.2 kb showed the high-
est homology with the T. subterraneum OASTL probe. In leaves,
OASTL mRNA showed similar levels in all conditions, in agree-
ment with the very low accumulation of As and metals in this
species; except for Pb + EDTA, where a slight increase was ob-
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Plant Biology 9 (2007) 7
Fig. 3 Northern blot analysis of OASTL from
legumes in response to As and heavy metals.
10 g total RNA from leaves or roots were
electrophoresed, blotted to Z-probe mem-
branes (Biorad), and hybridised sequentially
with an OASTL PCR fragment from Trifolium
subterraneum (this work), and the maize 17S
DNA for detection of 18S rRNA as a control of
total RNA quantity. Lanes C correspond to
RNA from plants grown in control conditions
in the absence of any As or heavy metals. (A)
Lotus japonicus, (B) Medicago sativa, (C) Trifoli-
um subterraneum.
served in parallel with the highest metal accumulation. In
roots of Lotus, two mRNA bands of very similar size were seen,
showing that the OASTL mRNA was also increased by Pb + EDTA
(Fig. 3 A). In Medicago sativa, a strong enhancement of OASTL
mRNA was observed in Cd and Pb, mainly in roots, in parallel
with the highest accumulation of these elements in the root
tissue, especially Pb, with the highest accumulation of this el-
ement in the presence of EDTA, whereas a decrease in the level
of transcript was seen with Cu (Fig. 3 B). In Trifolium subterra-
neum, (Fig. 3 C), the OASTL mRNA was increased by As, Cd, and
Pb, both in roots and leaves. The presence of Cu also produced
a decrease in the level of transcript in shoots, according to the
diminution in the level of OASTL protein bands in the presence
of Cu.
Discussion
Besides its role as the main plant macronutrient found in the
amino acids, Cys and Met, and in sulfolipids and coenzymes,
sulfur plays a critical role in many plant processes, including
redox cycles, metal detoxification, and secondary metabolism
(Hawkesford and De Kok, 2006). The main peptides involved
in plant metal resistance are phytochelatins and metallothio-
neins (Clemens, 2006; Pilon-Smits, 2005). In both molecules,
the proportion of the amino acid cysteine is very high (cysteine

8 Plant Biology 9 (2007)
is one of every two to three amino acids). Much evidence has
been obtained on the regulation of the enzymes involved in
phytochelatin biosynthesis by the presence of heavy metals
(Schfer et al., 1998; Xiang and Oliver, 1998). In particular,
O-acetylserine (thiol)lyase, the enzyme catalysing cysteine
biosynthesis from O-acetylserine and sulfide, has been shown
to be regulated by cadmium in Arabidopsis thaliana (Domn-
guez-Sols et al., 2001). Furthermore, overexpression of differ-
ent enzymes of the pathway led to an increase in metal resis-
tance and/or accumulation (Wawrzynskin et al., 2006; Sirko et
al., 2004; Gisbert et al., 2003; Zhu et al., 1999 a, b), although
some contradictory results have been obtained when over-
expression of phytochelatin synthase was attempted (Lee et
al., 2003).
Based on previous data showing that the overexpression of the
major cytosolic OASTL from Arabidopsis allowed the plant to
survive under severe heavy metal stress conditions (Domn-
guez-Sols et al., 2004), in this paper we studied the effect of
some heavy metals and metalloids on OASTL from legumes at
different levels, transcript and protein abundance and enzyme
activity. Our main aim was to obtain an insight into the poten-
tial of a model and two crop legumes in As and metal accumu-
lation. This was why relatively high concentrations of heavy
metals were used, in order to find a reference for the level of
tolerance of legumes to these toxic elements, as well as the
possibilities of using legumes in soil remediation. Legumes
are receiving increasing interest in soil regeneration due to
their known agronomic potential, good adaptation to poor
soils, and independence of nitrogen fertilisers. Several legumes
have been proposed in bioremediation experiments (Carrasco
et al., 2005; Pastor et al., 2003; Sriprang et al., 2002). Lotus ja-
ponicus was included in our study because it is now accepted
as a model legume, in which studies on many different aspects
have been conducted in laboratories worldwide (Pajuelo and
Stougaard, 2005). Besides this model legume, we also studied
OASTL regulation in two crop legumes belonging to different
genera, which have great potential in phytoremediation trials
due to high biomass production and existence of commercial
varieties of known agronomy value.
The enzyme activity increased in both shoots and roots of le-
gumes in the presence of Cd and Pb + EDTA. Among all the dif-
ferent OASTL isoforms detected by antibodies raised against
the A. thaliana major cytosolic OASTL, a low-molecular weight
isoform (32 kDa) is specifically enhanced in the presence of Cd
and Pb in Lotus japonicus and, particularly, in clover plants.
Further, the 1.2 kb mRNA level increases in response to Cd and
Pb + EDTA, particularly in roots, in parallel with a higher accu-
mulation of metals in the root tissue of this legume. As pro-
duced only a slight increase in expression in roots. However,
the presence of Cu produced a decrease in OASTL mRNA, pro-
tein, and activity levels. Copper detoxification in plants seems
to occur mainly via metallothioneins (Callahan et al., 2006;
Garca-Hernndez et al., 1998). In fact, overexpression of a pea
metallothionein in E. coli and plants led to an increase in toler-
ance to Cu, but not to other metals, such as Cd or Zn (Evans et
al., 1992). This could be a reason for the lack of a Cu effect on
the expression of the cytosolic OASTL, which is not directly
linked to metallothionein biosynthesis. In contrast, OASTL en-
zyme activity provides precursors for non-ribosomal synthesis
of PCs that are involved in As, Cd, and Pb detoxification (Cle-
mens, 2006). Furthermore, we do not know if the decrease in
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E. Pajuelo et al.
the level of OASTL expression in the presence of Cu is a specific
effect or a more general toxicity effect, since Cu was the most
toxic element tested at the concentrations used in this study,
on the basis of biomass production.
The potential of legumes in metal phytoextraction was ana-
lysed in a pot experiment under greenhouse conditions. Since
total phytoextraction depends on accumulation and total bio-
mass, both parameters were determined in legume plants
grown in the presence of As, Cd, Cu, and Pb + EDTA. With re-
gard to toxicity symptoms and biomass reduction, Cu was the
element that produced the most drastic biomass reduction (up
to 75%) and greater toxicity symptoms, at the concentrations
used. Lotus japonicus was more affected by treatment with
metals, whereas commercial varieties of alfalfa and clover
showed a less marked decrease in plant biomass, in response
to the presence of toxic elements. Lotus japonicus behaved as
an As excluder, with the minimum amount of As accumulated
in shoots (ratio of As accumulation in shoots to roots: 0.01). Al-
though, in terms of total phytoextraction, clover did accumu-
late a high amount of metals due to its higher plant biomass,
alfalfa had a higher accumulation of metal in shoots. This, to-
gether with the fact that biomass production of alfalfa was not
affected by Cd and Pb at the concentrations used in this experi-
ment, points to the suitability of this plant for Cd and Pb tol-
erance and accumulation. In the case of Pb, and due to the
presence of EDTA in the watering solution, there was a high ac-
cumulation in roots and a high translocation to aerial parts of
the plant. This effect could be observed in all legumes, espe-
cially in alfalfa. The amount of Pb accumulated in shoots is
over 2000 ppm, similar to the accumulation described for crop
plants proposed in phytoremediation (Bricker et al., 2004; Del
Ro et al., 2004; Gisbert et al., 2003). Much evidence has been
reported on the effect of chelates on plant phytoextraction, al-
ways resulting in an increase in metal uptake from soil and
metal translocation to the aerial parts of plants (Lombi et al.,
2001; Blaylock et al., 1997). However, in spite of the advan-
tages of using chelates in shoot metal accumulation, the addi-
tion of chelating agents in real in situ experiments must be
done under very controlled conditions to avoid the risk of
them leaching to groundwater (Cooper et al., 1999).
Taking all the above results together, we suggest a possible cor-
relation between the expression of OASTL in the presence of
metals and metal accumulation in legume plant tissues. Cu
produces a decrease in OASTL expression and is the most toxic
element, and Pb + EDTA causes the maximum OASTL activity,
protein and mRNA, particularly in roots, which have the maxi-
mum accumulation rate.
In this work, we have demonstrated that commercial varie-
ties of legumes can tolerate relatively high metal concentra-
tions, particularly Cu and Pb. Although legumes are clearly
non-hyperaccumulators, they could be considered in another
agronomic application: crop rotation with other remediative
plants. Because of the proliferation of weeds, predators, and
diseases that can cause significant yield reduction, crops, in-
cluding those used for soil remediation, must be rotated. For
longer term applications, as anticipated for most metal phy-
toextraction projects, it is unlikely that successful metal clean-
up can be achieved with only one remediative species in
monoculture, especially if multiple crops per year are project-
ed (Lasat, 2000). In this sense, legumes are widely used in crop
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Legume Phytoextraction Potentia Plant Biology 9 (2007) 9

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